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.

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.

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.

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.

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.

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.

Transcriptomic Changes in Zebrafish Embryos and Larvae Following Benzo[a]pyrene Exposure.

Benzo[a]pyrene (BaP) is an environmentally relevant carcinogenic and endocrine disrupting compound that causes immediate, long-term, and multigenerational health deficits in mammals and fish. Previously, we found that BaP alters DNA methylation patterns in developing zebrafish, which may affect gene expression. Herein, we performed a genome-wide transcriptional analysis and discovered differential gene expression and splicing in developing zebrafish. Adult zebrafish were exposed to control or 42.0 ± 1.9 µg/l BaP for 7 days. Eggs were collected and raised in control conditions or continuously exposed to BaP until 3.3 and 96 h post-fertilization (hpf). RNA sequencing (RNA-Seq) was conducted on zebrafish embryos and larvae. Data were analyzed to identify differentially expressed (DE) genes (changed at the gene or transcript variant level) and genes with differential exon usage (DEU; changed at the exon level). At 3.3 hpf, BaP exposure resulted in 8 DE genes and 51 DEU genes. At 96 hpf, BaP exposure altered expression in 1153 DE genes and 159 DEU genes. Functional ontology analysis by Ingenuity Pathway Analysis revealed that many disease pathways, including organismal death, growth failure, abnormal morphology of embryonic tissue, congenital heart disease, and adverse neuritogenesis, were significantly enriched for the DE and DEU genes, providing novel insights on the mechanisms of action of BaP-induced developmental toxicities. Collectively, we discovered substantial transcriptomic changes at the gene, transcript variant, and exon levels in developing zebrafish after early life BaP waterborne exposure, and these changes may lead to long-term adverse physiological consequences.

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.

Alteration in Pimephales promelas mucus production after exposure to nanosilver or silver nitrate.

The fish gill's ability to produce mucus effectively is a critical part of the stress response and protection against xenobiotic toxicity. Adult fathead minnows were exposed to silver nitrate (0.82 µg/L or 13.2 µg/L), polyvinylpyrrolidone-coated silver nanoparticles (11.1 µg/L or 208 µg/L), and citrate-coated silver nanoparticles (10.1 µg/L or 175 µg/L) for 96 h. Mucus concentrations based on glucose as a surrogate were determined at 0 h, 1 h, 2 h, 3 h, 4 h and 24 h after re-dosing each day. Higher mucus production rates following silver treatment were observed at the beginning as compared to controls and compared to after 3 d of exposure. Control fish produced consistent mucus concentrations throughout the exposure (0.62 mg/L and 0.40 mg/L at 24 h and 96 h, respectively). Following 24 h of exposure, all silver treatment groups produced significantly more mucus than controls. Following 96 h of exposure, mucus concentrations in treatment groups were significantly reduced compared with each respective treatment at 24 h. Reduced mucus production following long-term silver exposure could prevent the gills from removing silver, and thus increase toxicity.

Benzo[a]pyrene effects on reproductive endpoints in Fundulus heteroclitus.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that has been implicated in modulating aromatase enzyme function with the potential to interrupt normal reproductive function. The aim of this study was to use a fish model, Fundulus heteroclitus, to assess whether BaP exposure adversely impacts reproduction. Adult fish were exposed to waterborne BaP nominal concentrations of (0, 1, or 10 µg/l) for 28 days. Males and females were combined for the second half of the exposure (days 14-28) in order to quantitate egg production and fertilization success. Egg fertilization and subsequent hatching success of F1 embryos was significantly decreased by the high dose of BaP. In males, both gonad weight and plasma testosterone concentrations were significantly reduced compared to controls by 10 µg/l BaP. Histopathological examination of testes including spermatogonia, spermatocyte and spermatid cyst areas, percentage of cysts per phase, and area of spermatozoa per seminiferous tubule were not significantly affected. Other biomarkers, including male liver weight, liver vitellogenin (vtg) mRNA expression and sperm concentrations, were also not affected. In females, estradiol concentrations were significantly reduced after BaP exposure, but egg production, gonad weight, liver weight, vtg expression and oocyte maturation were not altered. Steroid concentrations in Fundulus larvae from exposed parents at 1 and 3 weeks posthatch were not significantly changed. BaP exposure at these environmentally relevant concentrations caused negative alterations particularly in male fish to both biochemical and phenotypic biomarkers associated with reproduction and multigenerational embryo survival.

Multigenerational effects of benzo[a]pyrene exposure on survival and developmental deformities in zebrafish larvae.

In the aquatic environment, adverse outcomes from dietary polycyclic aromatic hydrocarbon (PAH) exposure are poorly understood, and multigenerational developmental effects following exposure to PAHs are in need of exploration. Benzo[a]pyrene (BaP), a model PAH, is a recognized carcinogen and endocrine disruptor. Here adult zebrafish (F0) were fed 0, 10, 114, or 1,012 µg BaP/g diet at a feed rate of 1% body weight twice/day for 21 days. Eggs were collected and embryos (F1) were raised to assess mortality and time to hatch at 24, 32, 48, 56, 72, 80, and 96 h post fertilization (hpf) before scoring developmental deformities at 96 hpf. F1 generation fish were raised to produce the F2 generation followed by the F3 and F4 generations. Mortality significantly increased in the higher dose groups of BaP (2.3 and 20 µg BaP/g fish) in the F1 generation while there were no differences in the F2, F3, or F4 generations. In addition, premature hatching was observed among the surviving fish in the higher dose of the F1 generation, but no differences were found in the F2 and F3 generations. While only the adult F0 generation was BaP-treated, this exposure resulted in multigenerational phenotypic impacts on at least two generations (F1 and F2). Body morphology deformities (shape of body, tail, and pectoral fins) were the most severe abnormality observed, and these were most extreme in the F1 generation but still present in the F2 but not F3 generations. Craniofacial structures (length of brain regions, size of optic and otic vesicles, and jaw deformities), although not significantly affected in the F1 generation, emerged as significant deformities in the F2 generation. Future work will attempt to molecularly anchor the persistent multigenerational phenotypic deformities noted in this study caused by BaP exposure.

Global and gene specific DNA methylation changes during zebrafish development.

DNA methylation is dynamic through the life of an organism. Previous studies have primarily focused on DNA methylation changes during very early embryogenesis. In this study, global and gene specific DNA methylation in zebrafish (Danio rerio) embryos, larvae and adult livers were compared. The percent methylation of cytosines was low in 2 to 4.3h post fertilization (hpf) zebrafish embryos and was consistently higher in zebrafish older than 6 hpf. Furthermore, quantitative real-time PCR (qPCR) results showed relatively high DNA methyltransferase 1 (dnmt1) and low glycine N-methyltransferase (gnmt) mRNA expression in early embryogenesis. By studying methylation patterns and gene expression of five developmentally important genes, namely vasa, Ras-association domain family member 1 (rassf1), telomerase reverse transcriptase (tert), c-jun and c-myca, we found that the timing of changes in DNA methylation patterns was gene specific, and changes in gene expression were not necessarily correlated with the DNA methylation patterns.

Benzo[a]pyrene decreases global and gene specific DNA methylation during zebrafish development.

DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24µg/L from 2.5 to 96hpf to zebrafish embryos significantly decreased global cytosine methylation by 44.8% and promoter methylation in vasa by 17%. Consequently, vasa expression was significantly increased by 33%. In contrast, BaP exposure at environmentally relevant concentrations did not change CpG island methylation or gene expression in cancer genes such as ras-association domain family member 1 (rassf1), telomerase reverse transcriptase (tert), c-jun, and c-myca. Similarly, BaP did not change gene expression of DNA methyltransferase 1 (dnmt1) and glycine N-methyltransferase (gnmt). While total DNMT activity was not affected, GNMT enzyme activity was moderately increased. In summary, BaP is an epigenetic modifier for global and gene specific DNA methylation status in zebrafish larvae.

Expression of CYP1C1 and CYP1A in Fundulus heteroclitus during PAH-induced carcinogenesis.

CYP1C1 is a relatively newly identified member of the cytochrome P450 family 1 in teleost fish. However, CYP1C1's expression and physiological roles relative to the more recognized CYP1A in polycyclic aromatic hydrocarbons (PAHs) induced toxicities are unclear. Fundulus heteroclitus fry were exposed at 6-8 days post-hatch (dph) and again at 13-15dph for 6h to dimethyl sulfoxide (DMSO) control, 5mg/L benzo[a]pyrene (BaP), or 5mg/L dimethylbenzanthracene (DMBA). Fry were euthanized at 0, 6, 18, 24 and 30h after the second exposure. In these groups, both CYP1A and CYP1C1 protein expression were induced within 6h after the second exposure. Immunohistochemistry (IHC) results from fry revealed strongest CYP1C1 expression in renal tubular and intestinal epithelial cells. Additional fish were examined for liver lesions 8 months after initial exposure. Gross lesions were observed in 20% of the BaP and 35% of the DMBA-treated fish livers. Histopathologic findings included foci of cellular alteration and neoplasms, including hepatocellular adenoma, hepatocellular carcinoma and cholangioma. Strong CYP1A immunostaining was detected diffusely in altered cell foci and on the invading margin of hepatocelluar carcinomas. Lower CYP1A expression was seen in central regions of the neoplasms. In contrast, CYP1C1 was only detectable and highly expressed in proliferated bile duct epithelial cells. Our CYP1C1 results suggest the potential for tissue specific CYP1C1-mediated PAH metabolism but not a more chronic role in progression to liver hepatocellular carcinoma.

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.

Cytochrome P450-mediated 17beta-estradiol metabolism in zebrafish (Danio rerio).

Cytochrome P4501 (CYP1) and CYP3A proteins are primarily responsible for the metabolism of 17beta-estradiol (E(2)) in mammals. We have cloned and heterologously expressed CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP1D1, and CYP3A65 from zebrafish (Danio rerio) to determine the CYP-mediated metabolism of E(2) in a non-mammalian species. Constructs of each CYP cDNA were created using a leader sequence from the bacterial ompA gene to allow appropriate expression in Escherichia coli without 5' modification of the gene. Membrane vesicles were purified, and functional CYP protein was verified using carbon monoxide difference spectra and fluorescent catalytic assays with the substrates 7-ethoxyresorufin and 7-benzyloxy-4-(trifluoromethyl)-coumarin. Rates of in vitro E(2) metabolism into 4-hydroxyE(2) (4-OHE(2)), 2-hydroxyE(2) (2-OHE(2)), and 16alpha-hydroxyE(1) (16alpha-OHE(1)) metabolites were determined by gas chromatography/mass spectrometry. The 2-OHE(2) metabolite was produced by all CYPs tested, while 4-OHE(2) was only detected following incubation with CYP1A, CYP1B1, CYP1C1, and CYP1C2. The 16alpha-OHE(1) metabolite was only produced by CYP1A. The highest rates of E(2) metabolism were from CYP1A and CYP1C1, followed by CYP1C2. CYP1B1, CYP1D1, and CYP3A65 had low rates of E(2) metabolism. E(2) metabolism by zebrafish CYP1A, CYP1C1, and CYP1C2 produced similar ratios of 4-OHE(2) to 2-OHE(2) as previous studies with mammalian CYP1As. CYP1B1 formed the highest ratio of 4-OHE(2) to 2-OHE(2) metabolites. Contrary to mammals, these results suggest that fish CYP1A and CYP1C proteins are primarily responsible for E(2) metabolism, with only minor contributions from CYP3A65 and CYP1B1. Similar to mammals, 2-OHE(2) is the predominant metabolite from CYP-mediated E(2) metabolism in fish, suggesting that all vertebrate species produce the same major E(2) metabolite.

Benzo[a]pyrene effects on glycine N-methyltransferase mRNA expression and enzyme activity in Fundulus heteroclitus embryos.

Benzo[a]pyrene (BaP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) contaminant that is both a carcinogen and a developmental toxicant. We hypothesize that some of BaP's developmental toxicity may be mediated by effects on glycine N-methyltransferase (GNMT). GNMT is a mediator in the methionine and folate cycles, and the homotetrameric form enzymatically transfers a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine. SAM homeostasis, as regulated by GNMT, is critically involved in regulation of DNA methylation, and altered GNMT expression is associated with liver pathologies. The homodimeric form of GNMT has been suggested as the 4S PAH-binding protein. To further study BaP-GNMT interactions, Fundulus heteroclitus embryos were exposed to waterborne BaP at 10 and 100mug/L and both GNMT mRNA expression and enzyme activity were determined. Whole mount in situ hybridization showed GNMT mRNA expression was increased by BaP in the liver region of 7, 10 and 14dpf F. heteroclitus embryos. In contrast to mRNA induction, in vivo BaP exposure decreased GNMT enzyme activity in 4, 10 and 14dpf embryos. However, in vitro incubations of adult F. heteroclitus liver cytosol with BaP did not cause decreased enzyme activity. In conclusion, BaP exposure altered GNMT expression, which may represent a new target pathway for BaP-mediated embryonic toxicities and DNA methylation changes.

Benzo(a)pyrene induced glycine N-methyltransferase messenger RNA expression in Fundulus heteroclitus embryos.

Glycine N-methyltransferase (GNMT) is a mediator in the methionine and folate cycles, and is responsible for the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine. All the known DNA methyltransferases use SAM as a methyl donor thus, GNMT is critically involved in regulation of DNA methylation. Altered GNMT activities have been associated with liver pathologies including hepatocellular carcinoma. The homotetramer form of GNMT is enzymatically active, but the homodimeric form has been suggested as the 4S PAH-binding protein which may mediate CYP1A expression. To further understand the role of GNMT in benzo(a)pyrene (BaP)-related toxicity, full length Fundulus heteroclitus GNMT cDNA was cloned from adult liver. The open reading frame (ORF) of GNMT is 888 base pairs long and encodes a deduced protein of 295 amino acids which has 74% identity with human GNMT. Expression of GNMT mRNA was determined by quantitative RT-PCR. In unfertilized, 2days postfertilization (dpf), and 3 dpf embryos GNMT was constitutively higher than in 4, 7, 10 or 14 dpf embryos. Embryos were also exposed to waterborne BaP at 10 and 100 µg L⁻¹, and by 10 dpf the higher BaP dose caused increased expression of GNMT mRNA. These results suggest that PAH exposure may alter expression of an important physiological methylation mediator. Future work will be necessary to determine enzyme level effects of BaP exposure as well.