In the Swim of Cannabis: Developmental Toxicity and Metabolomic Pathway Alterations of Zebrafish Larvae Exposed to THC for the Assessment of Its Potential Environmental and Human Health Impact.

As the pharmacological properties and therapeutic applications of Cannabis sativa L. pace with the upsurge of interest of the scientific community in harnessing its constituent phytocannabinoids, illicit use may raise serious health issues. Tetrahydrocannabinol (THC) is one of the most well-known phytoactive constituents of cannabis and continues to garner scientific and public attention not only because of its pharmacological value but also because over-the-counter products of THC and prescription medications are becoming increasingly available from pharmacies, dispensaries, Internet, local retail stores, or by illicit means. Hence, a multidimensional approach was employed to examine the impact of THC on zebrafish larvae. The acute toxicity, expressed as LC50, was 1.54 mg/L. Adverse effects were observed on the phenotype, such as tail bending, pericardial edema, etc., even at concentrations lower than LC50, and fundamental functions of larvae (e.g., heart rate and cardiac contractility, and rhythm) were significantly affected. Behavioral changes were noticed, which were reflected in locomotor activity and sensitivity to light/dark changes. Finally, an untargeted metabolomic study was carried out to shed light on the metabolic alterations that occurred, providing substantiating evidence of the observed phenotype alterations. Overall, the potentially detrimental effects of THC on a vertebrate model are depicted.

The genetic population structure and temporal genetic stability of gilthead sea bream Sparus aurata populations in the Aegean and Ionian Seas, using microsatellite DNA markers.

We examined 662 gilthead sea bream Sparus aurata from wild samples of the species in the Aegean and Ionian Seas, using 20 EST-linked microsatellite markers, in three multiplex panels, as well as seven anonymous loci. Most of the markers were revealed to be highly polymorphic. We found low genetic differentiation between the sampling stations/areas with total FST 0.002 (P < 0.05). Based on comparison of five temporal samples, our results indicate genetic data consistency over time for all tested samples, pointing to stable populations, despite reported repeated escape events. Our results confirm the genetic population structure previously observed in these specific areas, using by far more markers than in previous studies in both coding and non-coding DNA loci. The limited genetic structure and the temporal genetic stability indicate neither major genetic differentiation of local populations by geographic isolation nor influence from anthropogenic factors. These results provide a baseline for future reference in any management programme of both wild and farmed population of S. aurata as well as of other aquaculture species with a potential introgression among farmed and wild populations.

N-(p-coumaroyl) serotonin inhibits glioblastoma cells growth through triggering S-phase arrest and apoptosis.

Glioblastoma is the most common and most malignant primary brain tumor with a median survival of 15 months. N-(p-coumaroyl) serotonin (CS) is an indole alkaloid with antioxidant, cardioprotective effects after ischemia and antitumor activity. In the present study we sought to determine whether could exert cytotoxic and cytostatic effects in glioma cells in vitro. CS was tested for toxicity in zebrafish. We investigated the effect of CS in U251MG and T98G glioblastoma cell lines. Viability and proliferation of the cells were examined with trypan blue exclusion assay and the xCELLigence system. Cell cycle, activation of caspase-8, mitochondrial membrane potential and CD24/CD44/CD56/CD15/CD71 expression were tested with flow cytometry. Treatment with CS significantly reduced cell viability in both cell lines tested. Induction of cell death and cell cycle arrest at G2/M and S-phase was confirmed with flow cytometry in both cell lines. CS produced significant higher activity of caspase-8 compared to control. After treatment with CS there was a dose-dependent increase in CD15 and CD71 expression, whereas there was no change in CD24/CD44/CD56 expression in both cell lines. The zebrafish mortality on the fifth post fertilization day was zero for even 1 mM of CS concentration. The treatment of glioblastoma cell lines with CS may represent a novel strategy for targeting glioblastoma. Further studies are obviously needed to elucidate the complete mechanism of its antitumor activity.

Transcriptional and Behavioral Responses of Zebrafish Larvae to Microcystin-LR Exposure.

Microcystins are cyclic heptapeptides that constitute a diverse group of toxins produced by cyanobacteria. One of the most toxic variants of this family is microcystin-LR (MCLR) which is a potent inhibitor of protein phosphatase 2A (PP2A) and induces cytoskeleton alterations. In this study, zebrafish larvae exposed to 500 µg/L of MCLR for four days exhibited a 40% reduction of PP2A activity compared to the controls, indicating early effects of the toxin. Gene expression profiling of the MCLR-exposed larvae using microarray analysis revealed that keratin 96 (krt96) was the most downregulated gene, consistent with the well-documented effects of MCLR on cytoskeleton structure. In addition, our analysis revealed upregulation in all genes encoding for the enzymes of the retinal visual cycle, including rpe65a (retinal pigment epithelium-specific protein 65a), which is critical for the larval vision. Quantitative real-time PCR (qPCR) analysis confirmed the microarray data, showing that rpe65a was significantly upregulated at 50 µg/L and 500 µg/L MCLR in a dose-dependent manner. Consistent with the microarray data, MCLR-treated larvae displayed behavioral alterations such as weakening response to the sudden darkness and hypoactivity in the dark. Our work reveals new molecular targets for MCLR and provides further insights into the molecular mechanisms of MCLR toxicity during early development.

Metabolomic Fingerprint Assay in Zebrafish Embryos.

In order for new drugs to enter the market, extensive studies are needed to examine toxic effects. Among others, teratogenicity studies are of paramount importance. Of even higher importance is to gain knowledge on the biological responses that take place upon drug exposure, so as to have a better understanding of the molecular mechanisms that govern developmental changes. Metabolomics is the research field that studies the changes in the chemical composition of metabolites contained within cells. Conducting metabolomics studies results in valuable information. Zebrafish is a vertebrate model organism that bridges in vivo assays and in vivo studies. In this chapter, we propose a metabolomic fingerprint assay for the study of metabolic changes in zebrafish embryos upon exposure to various drugs. The metabolome of zebrafish is extracted, and the 1H-NMR spectrum is recorded. Using open-access metabolomic databases, a list of tentative metabolites is retrieved. The presence of the tentative metabolites is further confirmed by UHPLC-HRMS. Ultimately, after a metabolic pathway analysis, the metabolic network is revealed and useful conclusions can be drawn.

Glioblastoma research on zebrafish xenograft models: a systematic review.

Glioblastoma (GBM) constitutes the most common primary brain tumor in adults. The challenges in GBM therapeutics have shed light on zebrafish used as a promising animal model for preclinical GBM xenograft studies without a standardized methodology. This systematic review aims to summarize the advances in zebrafish GBM xenografting, compare research protocols to pinpoint advantages and underlying limitations, and designate the predominant xenografting parameters. Based on the PRISMA checklist, we systematically searched PubMed, Scopus, and ZFIN using the keywords "glioblastoma," "xenotransplantation," and "zebrafish" for papers published from 2005 to 2022, available in English. 46 articles meeting the review criteria were examined for the zebrafish strain, cancer cell line, cell labeling technique, injected cell number, time and site of injection, and maintenance temperature. Our review designated that AB wild-type zebrafish, Casper transparent mutants, transgenic Tg(fli1:EGFP), or crossbreeding of these predominate among the zebrafish strains. Orthotopic transplantation is more commonly employed. A number of 50-100 cells injected at 48 h post-fertilization in high density and low infusion volume is considered as an effective xenografting approach. U87 cells are used for GBM angiogenesis studies, U251 for GBM proliferation studies, and patient-derived xenograft (PDX) to achieve clinical relevance. Gradual acclimatization to 32-33 °C can partly address the temperature differential between the zebrafish and the GBM cells. Zebrafish xenograft models constitute valuable tools for preclinical studies with clinical relevance regarding PDX. The GBM xenografting research requires modification based on the objective of each research team. Automation and further optimization of the protocol parameters could scale up the anticancer drug trials.

Morphotype induced changes in the life history and population dynamics of an hippolytid shrimp.

One of the most exceptional, loud paradigm of shape polymorphisms constitutes the "rostral loss" condition in hippolytid shrimps. The intertidal shrimp Hippolyte sapphica includes two conspecific morphotypes, one of which demonstrates a neotenic rostrum (morph-B). Morphs' rostral elongation is controlled by a single genetic locus, with long rostra (morph-A) representing the recessive state and short, larval-like ones the completely dominant state. Geometrics morphometry on the species morphotypes revealed also the homozygous/heterozygous state of the gene site along with some induced body' adaptations, which compete the micro-evolutionary disadvantage of the "rostral loss". We found recently that females' viability and maternal energy investment selectively favors morph-A. The present contribution detects and discuss comparatively demographic and reproductive traits in species mixed (both morphs) and unmixed populations. Our results show that this sharp dimorphic rostral condition is a sex-related marker and that the species is gonochoric. Presence of morph-B results to (a) lower egg production (b) higher seasonally males' percentage (c) morph-A females' earlier maturation and (d) higher fecundity in morph-A mixed populations. It seems that the "rostral loss" state induces complex adaptations between the two morphotypes through sex ratios equilibria, morphotypes' growth rates, and morphs' fecundity differentiate inputs throughout the seasons.

Assessment of microcystin distribution and biomagnification in tissues of aquatic food web compartments from a shallow lake and evaluation of potential risks to public health.

The objectives of this study were: (1) to examine the distribution and bioaccumulation of microcystins in the main components of the food web (phytoplankton, zooplankton, crayfish, shrimp, mussel, snail, fish, frog) of Lake Pamvotis (NW Greece), (2) to investigate the possibility of microcystin biomagnification and (3) to evaluate the potential threat of the contaminated aquatic organisms to human health. Significant microcystin concentrations were detected in all the aquatic organisms during two different periods, with the higher concentrations observed in phytoplankton and the lower in fish species and frogs. This is the first study reporting microcystin accumulation in the body of the freshwater shrimp Atyaephyra desmsaresti, in the brain of the fish species common carp (Cyprinus carpio) and in the skin of the frog Rana epirotica. Although there was no evidence for microcystin biomagnification, the fact that microcystins were found in lake water and in the tissues of aquatic organisms, suggests that serious risks to animal and public health are possible to occur. In addition, it is likely to be unsafe to consume aquatic species harvested in Lake Pamvotis due to the high-concentrations of accumulated microcystins.

Detection of microcystins in Pamvotis lake water and assessment of cyanobacterial bloom toxicity.

Lake Pamvotis is a shallow, eutrophic Mediterranean lake with ecological significance. This paper deals with the evaluation of cyanobacterial toxicity in Lake Pamvotis. ELISA and HPLC revealed the presence of significant amounts of MCYST-LR. Danio rerio bioassay confirmed the toxic nature of the bloom. Cyanobacterial extracts had adverse toxic effects on development of D. rerio. Also, it was shown that cyanobacterial extracts containing environmentally detected concentrations of MCYST can cause reduced survival rate of fish species. The results clearly indicate that cyanobacterial blooms in Lake Pamvotis may be regarded as human and fish health hazard. Continuous monitoring of the lake is suggested, in order to prevent future possible intoxications.

DNA barcoding identification of Greek freshwater fishes.

Biodiversity is a key factor for the functioning and efficiency of an ecosystem. Greece, though covering a relatively small surface area, hosts a great deal of species diversity. This is especially true for freshwater fishes. In recent years, the traditional methods of species identification have been supplemented by the use of molecular markers. The present study therefore aims to extensively produce DNA barcodes for Greek freshwater fish species and investigate thoroughly if the presently accepted species classification is in agreement with molecular data. A 624-bases long fragment of the COI gene was sequenced, from 406 freshwater fish specimens belonging to 24 genera and originating from 18 lake and river sites. These sequences were used along with 596 sequences from the same genera, recovered from BOLD, for the construction of phylogenetic trees and the estimation of genetic distances between individuals. In total, 1002 sequences belonging to 72 species were analyzed. The method was found to be effective for 55 of 72 studied species. 17 closely related species with low interspecific genetic distances were observed, for which further study is proposed. It should also be noted that, in four cases, cryptic diversity was observed, where groups originally identified as one species exhibited genetic distance great enough to be separated into discrete species. Region specific haplotypes were also detected within populations of 14 species, giving the possibility to identify even the geographic origin of a species. Our findings are discussed in the light of the rich history of the Balkan peninsula and provide a significant steppingstone for the further study of Greek and European freshwater fish biodiversity.

Metabolomic Profiling Unveils the Impact of Non-Doped and Heteroatom-Doped Carbon Nanodots on Zebrafish (Danio rerio) Embryos.

Recently, concern has been raised over the transport, transformation, and fate of carbon nanodots (CNDs) after their release into the environment. Their toxicity towards organisms and humans has recently been addressed as an important issue. In this study, a metabolomic approach was employed to obtain an insight into the effect of CNDs (either pristine or doped with nitrogen and nitrogen/sulfur) on zebrafish. Embryos were exposed to concentrations corresponding to lethal concentration (LC) LC50 (550, 400, and 150 µg mL-1), LC50/2 (275, 200, and 75 µg mL-1), and LC50/4 (138, 100, and 38 µg mL-1) of the three CNDs (non-doped, N-doped, and N,S-codoped, respectively) to scrutinize the interactions of the CNDs with the larvae. Numerous differences in the metabolic pathways were recorded in all cases. Seven metabolic pathways were detected in the control larvae. When the larvae were exposed to concentrations equal to LC50, LC50/2, and LC50/4 of non-doped CNDs, 12, 12, and 3 metabolic pathways were detected, respectively. In the case of N-doped CNDs, 4, 7, and 4 pathways were detected, while in the case of N,S-codoped CNDs, 8, 5, and 5 pathways were detected when exposed to concentrations of LC50, LC50/2, and LC50/4, respectively. In all cases, certain metabolic pathways were altered while others were either down-regulated or up-regulated. Some of these changes include the activation of alanine, aspartate, and glutamate metabolism, aminoacyl-tRNA biosynthesis, butanoate metabolism, D-glutamine, and D-glutamate metabolism, glutathione metabolism, selenoamino acid metabolism, valine, leucine, and isoleucine degradation pathways. Moreover, the deactivation of starch and sucrose metabolism, the glycine, serine, and threonine metabolism, among others, were recorded. Our findings underline the importance to further study the impact of CNDs on marine organisms. As zebrafish has been shown to share many similarities with humans in bioprocesses and genome, it can be assumed that CNDs may also pose a threat to human health.

Curcumin and Radiotherapy Exert Synergistic Anti-Glioma Effect In Vitro.

Curcumin, a bioactive polyphenol, is known to have anticancer properties. In this study, the effectiveness of curcumin pretreatment as a strategy for radio-sensitizing glioblastoma cell lines was explored. For this, U87 and T98 cells were treated with curcumin, exposed to 2 Gy or 4 Gy of irradiation, and the combined effect was compared to the antiproliferative effect of each agent when given individually. Cell viability and proliferation were evaluated with the trypan blue exclusion assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The synergistic effects of the combination treatment were analyzed with CompuSyn software. To examine how the co-treatment affected different phases of cell-cycle progression, a cell-cycle analysis via flow cytometry was performed. Treatment with curcumin and radiation significantly reduced cell viability in both U87 and T98 cell lines. The combination treatment arrested both cell lines at the G2/M phase to a higher extent than radiation or curcumin treatment alone. The synergistic effect of curcumin when combined with temozolomide resulted in increased tumor cell death. Our results demonstrate for the first time that low doses of curcumin and irradiation exhibit a strong synergistic anti-proliferative effect on glioblastoma cells in vitro. Therefore, this combination may represent an innovative and promising strategy for the treatment of glioblastoma, and further studies are needed to fully understand the molecular mechanism underlying this effect.

Accumulation of microcystins in water and fish tissues: an estimation of risks associated with microcystins in most of the Greek Lakes.

Toxin-producing cyanobacteria in lakes and reservoirs form a threat to humans as well as various forms of aquatic life. This study is an investigation into the occurrence and distribution of Microcystins (MCYST) in 13 Greek Lakes. The distribution of MCYST in water and surface scum and toxin bioaccumulations in the omnivorous fish species Carassius gibelio were surveyed in all lakes. Considerable amounts of MCYST were found in water and scum of all lakes, irrespective of the trophic state, the type of the lake, and the reported dominant cyanobacterial species. Toxin accumulation in six tissues (liver, brain, intestine, kidney, ovary, and muscle) of C. gibelio was also analyzed. Even though the target organ for MCYST is the liver, in our study, MCYST were found also in the rest of C. gibelio tissues in the following order: liver > intestine > kidney > brain > ovaries > muscle. Risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCYST. Our findings suggest that the amounts of MCYST found in water of Lakes Kastoria, Koronia, Pamvotis, Doirani, Mikri Prespa, Petron, and Zazari, pose adverse health risks. Also, it is likely to be unsafe to consume C. gibelio in Lakes Koronia, Kastoria, Pamvotis, and Mikri Prespa due to the high concentrations of accumulated MCYST.

Assessing the zooplankton community and environmental factors in a Mediterranean wetland.

Mediterranean wetlands represent unique repositories of biodiversity, but these ecosystems are increasingly threatened by human-induced habitat loss. Seventy percent of Greek wetlands (ponds, mires, marshes, etc.) have been lost in the past 80 years due to human intervention. In Greece habitat types of mires, listed in Directive 92/43/EEC, have been recorded in a few locations, one of the most important is Kalodiki wetland. Eutrophication key elements were determined at four sampling stations throughout 1 year in order to monitor the trophic conditions. Moreover, the zooplankton community was described as biological element relevant in the assessment of the ecological status of Kalodiki wetland. Kalodiki wetland exhibits nutrient concentrations corresponding to eutrophic conditions while according to chlorophyll-a values it is classified between mesotrophic and eutrophic status depending mostly on the sampling period. As concerning zooplankton community, it appears poor in species and dominated by small-sized organisms, which is generally typical of eutrophic, disturbed systems. Differences among zooplankton assemblages over seasons as well as among sampling sites highlight the role of both abiotic and biotic factors.

Induced toxicity in early-life stage zebrafish (Danio rerio) and its behavioral analysis after exposure to non-doped, nitrogen-doped and nitrogen, sulfur-co doped carbon quantum dots.

In this study, the effects of doping of CQDs with alternative functional groups (dopants) were evaluated through embryonic development of zebrafish (Danio rerio). The CQDs were synthesized using simple and low-cost sources: Non-doped (citric acid was used as the carbon source), nitrogen-doped (N-doped) and nitrogen, sulfur-co-doped (N,S-doped). The CQDs induced significant toxicity to zebrafish (>150 µg/mL) and the toxic effects were dose-dependent. The N,S-doped CQDs were the most toxic (LD50 = 149.92 µg/mL), followed by the N-doped CQDs (LD50 = 399.95 µg/mL) while the non-doped CQDs were the least toxic (LD50 = 548.48 µg/mL) of the three. The growth rate (GR) was affected following the toxicity pattern (GRNS-doped

Cannabinol in the spotlight: Toxicometabolomic study and behavioral analysis of zebrafish embryos exposed to the unknown cannabinoid.

Cannabinol (CBN) is a degradation product of the cannabis metabolite Δ9-tetrahydrocannabinol. The CBN concentration in cannabis leaves ranges between 0.1 and 1.6% (w/w of dry weight); it increases as the plant ages and its formation is affected by the storage conditions. As CBN has not been extensively studied so far, the need to examine its impact in vivo is imperative due to the increasing use of cannabis globally. In the study herein, the CBN toxicity, effects on heart physiology, morphological malformations, behavioral changes and alterations in metabolic pathways of zebrafish larvae upon CBN exposure to sublethal concentrations were examined. The LD50 value was estimated at 1.12 mg/l. At the same time, malformations in zebrafish larvae increased significantly in a dose-dependent manner and exposure to CBN concentrations greater than 0.75 mg/l provoked abnormalities like pericardial edema, yolk sac anomalies and tail bending. Concentrations above this threshold resulted in elongated and shorter in width hearts and in separation of ventricle from atrium. The total movement distance and velocity were increased in dark and decreased in light conditions, in a concentration-dependent manner. Our results showed that CBN acts both as a stimulant and a sedative, with larvae to exhibit altered velocity and bradycardia, respectively. The metabolomic analysis revealed alterations mainly to amino acids, which are related to acute toxicity and hint towards systemic metabolic and neuropathophysiological changes. Taken together, our data indicate increased toxic effects as CBN exposure concentration increases, which should be taken into consideration when studying the impact of cannabis on organisms.

Deglucohellebrin: A Potent Agent for Glioblastoma Treatment.

BACKGROUND: Glioblastoma is the most common primary brain tumor in adults with a dismal prognosis. To date, several anticancer agents have been isolated from plants. Helleborus odorus subsp. Cyclophyllus is an endemic plant of the Balcan flora. Herewith, we investigated for the first time, the anti-glioma effect of deglucohellebrin (DGH) extracted from the roots of Helleborus. METHODS: We investigated the effect of DGH in U251MG, T98G and U87G glioblastoma cell lines. We selected the T98G cells because of their inherent temozolomide resistance. RESULTS: The IC50 value of reduced viability for DGH was 7x10-5M in U251MG cells, 5x10-5M for the T98G cells and 4x10-5M in U87G cells during 72h treatment. DGH induced G2/M cell cycle arrest, caspace-8 activation and significant mitochondrial membrane depolarization, suggesting the activation of the intrinsic, mitochondrial- dependent apoptotic pathway. DGH and temozolomide induced changes in CDs' expression in U251MG and T98G cells. In zebrafish, DGH did not induce toxicity or behavioral alterations. CONCLUSION: The present study is the first to determine the anti-glioma activity of DGH. DGH may be a potent agent for glioblastoma treatment and further studies are needed.

Typology, classification and management issues of Greek lakes: implication of the Water Framework Directive (2000/60/EC).

The European Water Framework Directive 2000/60/EC, requires the determination of ecological status in European waterbodies, based primarily on biological indicators, and that minimum good ecological quality is obtained by 2015. We used background morphometric, hydrologic, physico-chemical and biological data, in order to assess the typology of the main Greek lakes. Correlation analysis and multivariate analysis (Principal Component Analysis) were applied for the data statistical elaboration. Lakes were classified according to the characteristics suggested in the Directive and specified in the Annex II. Statistical analysis revealed significant correlations between the lake variables as well as the grouping of the Greek lakes according to their abiotic characteristics. Lake water uses, in most cases competed each other, were identified and management practices were also recorded. We suggest that the future conservation efforts should focus on a new holistic management philosophy taking into consideration the catchment's management.

Speciation of phosphorus fractionation in river sediments by explanatory data analysis.

This article investigates the controls on sediment phosphorus (P) speciation dynamics as a function of its fractionation into chemically defined operational pools along a river continuum. A total of 27 variables were analyzed in bed sediment samples collected for one year from six sampling points, along a 75km river continuum (Louros River, NW Greece). Multivariate explanatory analysis of the complex experimental data matrix was performed in order to unravel the spatial pattern of P speciation. Non-parametric examinations were also applied in order to elucidate the temporal variations encountered in the speciation of P. The results suggest that inorganic P species control P bioavailability in space and time. Organic P was found to be very reactive among the various fractions thus changing its bioavailability but exhibited no temporal variation. The utility of the proposed approach in the differentiation of natural and anthropogenic P inputs and their classification to point and non-point sources is demonstrated presenting a significant improvement compared to mere fractionation analysis.

Host-parasite relationships: occurrence and effect of the parasitic isopod Mothocya epimerica on sand smelt Atherina boyeri in the Mesolongi and Etolikon Lagoons (W. Greece).

The parasite-host relationship between Mothocya epimerica, Costa 1851 (Isopoda: Flabellifera: Cymothoidae) and sand smelt Atherina boyeri (Osteichthyes: Atherinidae) fish populations were studied in the Mesolongi and Etolikon Lagoons (W. Greece). Prevalence varied during the year from 12.5% in November to 52.5% in September; overall mean prevalence was 41.9%. Parasite size increased with host size. Infections did not have a significant effect on the host's body condition, such as length-weight relationship, gonadosomatic index, hepatosomatic index and relative condition factor. Histological damage to the host was observed on the gills (especially in the second and third and arches upon which the female parasite rests her abdomen). The physiological cost resulting from this infection seems to be little and probably does not constitute a serious threat for individual host survival.