Occurrence and human health risk assessment of mineral elements and pesticides residues in bee pollen.

Bee pollen contains a diversity of bioactive components. Nevertheless, since pollen is retrieved from a variety of plants, including the cultivated ones which are subjected to agrochemical treatments, its contamination is unavoidable. In this context, 45 samples of pollen were analysed with optimized analytical methods for trace and macro elements (ICP-MS), pesticides and metabolites residues (LC & GC-MS/MS) content. According to the results, potassium and iron were the most abundant in terms of concentration and frequency of detection, while the contribution of the most hazardous elements, such as lead, arsenic, cadmium and mercury, to the total concentration of trace elements was lower than 1%. For pesticides, coumaphos was the most frequently detected in the examined samples (22%), followed by propargite, azoxystrobin, dimethoate and cypermethrin. Non-carcinogenic health risk assessment demonstrated in the majority of cases negligible risk for adults and children. On the contrary, carcinogenic risk assessment considering a worst case scenario disclosed nickel and in less extent chromium and arsenic, as risk drivers, exhibiting in several samples carcinogenic risk values for adults above the safety threshold. Yet, regarding that both adults and children unlikely will daily consume such pollen quantities, especially on a long-term basis, an overestimation of risk should be appraised.

Macro and Trace Elements in Hemp (Cannabis sativa L.) Cultivated in Greece: Risk Assessment of Toxic Elements.

The accumulation of hazardous contaminants in Cannabis sativa L. raises warning signs regarding possible adverse effects on human health due to the consumption of herbal medicines and/or other herbal edible products made from cannabis. Thus, there is an urge to investigate the levels of hazardous contaminants, such as heavy metals, in cannabis plant. In the present study, 29 macro and trace elements, including both beneficial and toxic elements (heavy metals and metalloids), were investigated in 90 samples of Cannabis sativa L. collected from Greece. According to the results, the detected concentrations of macro elements in the leaves/flowers of cannabis ranged between 28 and 138,378 ppm, and of trace elements between 0.002 and 1352.904 ppm. Although the concentrations of elements varied among the samples, their accumulation pattern was found to be similar, with the contribution of toxic elements to the total concentration of trace elements being below 1%. The detected levels of the most toxic elements were below the prescribed limits established by the WHO, while the calculated THQ and CR values showed no risk (non-carcinogenic and carcinogenic) for the population exposed to the current cannabis samples. Positive correlation between the concentration of elements and cannabis geographical origin and variety was observed. Cannabis leaves/flowers were more contaminated with trace and macro elements than seeds.

Pesticides residues and metabolites in honeybees: A Greek overview exploring Varroa and Nosema potential synergies.

The aim of this study was to investigate reported cases of honeybee mortality incidents and the potential association to pesticide exposure and to their metabolites. The same honeybee samples were also assessed for Varroa mites, and Nosema microsporidia provoked infections to provide an integrated picture of all observable stressors that may impact bees' survival. Thus, honeybee samples from different areas of Greece (2014-2018) were analyzed for the presence of pesticide residues and metabolites. In this context, an existing LC-ESI-QqQ-MS multiresidue method of analytes of different chemical classes such as neonicotinoids, organophosphates, triazoles, carbamates, was enriched with additional active substances, developed and validated. A complementary GC-EI-QqQ-MS method was also exploited for the same scope covering pyrethroid compounds. Both methods monitored more than 150 active substances and metabolites and presented acceptable linearity over the ranges assayed. The calculated recoveries ranged from 65 to 120% for the three concentration levels, while the precision (RSD%) values ranged between 4 and 15%. Therefore, this approach proved sufficient to act as a monitoring tool for the determination of pesticide residues in cases of suspected honeybee poisoning incidents. From the analysis of 320 samples, the presence of 70 active substances and metabolites was confirmed with concentrations varying from 1.4 ng/g to 166 µg/g. Predominant detections were the acaricide coumaphos, several neonicotinoids exemplified by clothianidin, organophosporous compounds dimethoate and chlorpyrifos, and some pyrethroids. Metabolites of imidacloprid, chlorpyrifos, coumaphos, acetamiprid, fenthion and amitraz were also identified. Concerning Nosema and Varroa they were identified in 27 and 22% of samples examined, respectively, verifying their prevalence and coexistence with pesticides and their metabolites in honeybees.

A Pesticide Residues Insight on Honeybees, Bumblebees and Olive Oil after Pesticidal Applications against the Olive Fruit Fly Bactrocera oleae (Diptera: Tephritidae).

In 2017 and 2018, a field survey was initiated on Greek olive orchards to investigate the attractiveness of bait spray applications and the impact of cover and bait sprays applied against the olive fruit fly Bactrocera oleae (Diptera: Tephritidae), on the honeybee, Apis mellifera L. and bumblebees Bombus terrestris, by investigating the pesticides' residual prevalence. Bee colonies were evenly distributed in three sites located on coastal areas of Western Crete and visited almost weekly between July and October. Samples collected, were analyzed using existing or developed-optimized liquid and gas chromatographic methods. In bee samples, concentrations varied from 0.0013 to 2.3 mg/kg for dimethoate, from 0.0013-0.059 mg/kg for its metabolite omethoate, and from 0.0035 to 0.63 mg/kg regarding the pyrethroids, ß-cyfluthrin and λ-cyhalothrin. In one bee sample dimethoate concentration exceeded both acute oral and contact median lethal dose (LD50). Residue findings in bees, along with verified olive oil residues corroborated that those insecticides had been applied in the olive orchards and transferred to bees. The possibility of non-target effects of the bait sprays to the bees, as well as the impact of the contaminated olive to the bees are discussed.

An essay on ecosystem availability of Nicotiana glauca graham alkaloids: the honeybees case study.

BACKGROUND: Invasive plant species pose a significant threat for fragile isolated ecosystems, occupying space, and consuming scarce local resources. Recently though, an additional adverse effect was recognized in the form of its secondary metabolites entering the food chain. The present study is elaborating on this subject with a specific focus on the Nicotiana glauca Graham (Solanaceae) alkaloids and their occurrence and food chain penetrability in Mediterranean ecosystems. For this purpose, a targeted liquid chromatography electrospray tandem mass spectrometric (LC-ESI-MS/MS) analytical method, encompassing six alkaloids and one coumarin derivative, utilizing hydrophilic interaction chromatography (HILIC) was developed and validated. RESULTS: The method exhibited satisfactory recoveries, for all analytes, ranging from 75 to 93%, and acceptable repeatability and reproducibility. Four compounds (anabasine, anatabine, nornicotine, and scopoletin) were identified and quantified in 3 N. glauca flowers extracts, establishing them as potential sources of alien bio-molecules. The most abundant constituent was anabasine, determined at 3900 µg/g in the methanolic extract. These extracts were utilized as feeding treatments on Apis mellifera honeybees, resulting in mild toxicity documented by 16-18% mortality. A slightly increased effect was elicited by the methanolic extract containing anabasine at 20 µg/mL, where mortality approached 25%. Dead bees were screened for residues of the N. glauca flower extracts compounds and a significant mean concentration of anabasine was evidenced in both 10 and 20 µg/mL treatments, ranging from 51 to 92 ng/g per bee body weight. Scopoletin was also detected in trace amounts. CONCLUSIONS: The mild toxicity of the extracts in conjunction with the alkaloid and coumarin residual detection in bees, suggest that these alien bio-molecules are transferred within the food chain, suggesting a chemical invasion phenomenon, never reported before.

Novel Carbonyl Analogs of Tamoxifen: Design, Synthesis, and Biological Evaluation.

Aim of this work was to provide tamoxifen analogs with enhanced estrogen receptor (ER) binding affinity. Hence, several derivatives were prepared using an efficient triarylethylenes synthetic protocol. The novel compounds bioactivity was evaluated through the determination of their receptor binding affinity and their agonist/antagonist activity against breast cancer tissue using a MCF-7 cell-based assay. Phenyl esters 6a,b and 8a,b exhibited binding affinity to both ERα and ERß higher than 4-hydroxytamoxifen while compounds 13 and 14 have shown cellular antiestrogenic activity similar to 4-hydroxytamoxifen and the known ER inhibitor ICI182,780. Theoretical calculations and molecular modeling were applied to investigate, support and explain the biological profile of the new compounds. The relevant data indicated an agreement between calculations and demonstrated biological activity allowing to extract useful structure-activity relationships. Results herein underline that modifications of tamoxifen structure still provide molecules with substantial activity, as portrayed in the inhibition of MCF-7 cells proliferation.

Assessment of field re-entry exposure to pesticides: A dislodgeable foliar residue study.

A dislodgeable foliar residue study was conducted in greenhouse pepper and tomato on the island of Crete, Greece, following the spray application of an SC insecticide (with active substance (a.s.) tebufenozide) and an EC fungicide (a.s. bupirimate). Furthermore, for the assessment of worker exposure to pesticides - as a result of re-entering the treated crops - a worker dermal exposure study was carried out during the tasks of tying or pruning, which allowed the transfer coefficient values for the specific tasks to be determined. Pesticide residues were analysed with an in house developed and fully validated HPLC-ESI/MS analytical method. The results from the study resulted in transfer coefficient values which were in agreement with current EFSA guideline values in most of the cases with the exception of bupirimate in a tomato greenhouse. In that case, high potential dermal exposure and low dislodgeable foliar residue values were observed, which is thought to be due to the moist leaves collected during sampling and monitoring, which led to greater than expected transfer coefficient values.

Revisiting Greek Propolis: Chromatographic Analysis and Antioxidant Activity Study.

Propolis is a bee product that has been extensively used in alternative medicine and recently has gained interest on a global scale as an essential ingredient of healthy foods and cosmetics. Propolis is also considered to improve human health and to prevent diseases such as inflammation, heart disease, diabetes and even cancer. However, the claimed effects are anticipated to be correlated to its chemical composition. Since propolis is a natural product, its composition is consequently expected to be variable depending on the local flora alignment. In this work, we present the development of a novel HPLC-PDA-ESI/MS targeted method, used to identify and quantify 59 phenolic compounds in Greek propolis hydroalcoholic extracts. Amongst them, nine phenolic compounds are herein reported for the first time in Greek propolis. Alongside GC-MS complementary analysis was employed, unveiling eight additional newly reported compounds. The antioxidant activity study of the propolis samples verified the potential of these extracts to effectively scavenge radicals, with the extract of Imathia region exhibiting comparable antioxidant activity to that of quercetin.

Effects of peppermint tea consumption on the activities of CYP1A2, CYP2A6, Xanthine Oxidase, N-acetyltranferase-2 and UDP-glucuronosyltransferases-1A1/1A6 in healthy volunteers.

Peppermint leaves are widely used for the symptomatic treatment of digestive disorders. Previous studies have shown significant effects of its natural products on human enzyme activity; however, there is no study available concerning the effects of peppermint tea on metabolizing enzymes in humans. Aim of the present study was to investigate the effect of peppermint tea on CYP1A2, CYP2A6, Xanthine Oxidase (XO), N-acetyltranferase-2 (NAT2) and UDP-glucuronosyltransferases-1A1/1A6 (UGT1A1/1A6) activities in healthy subjects. Four males and five females consumed peppermint tea (2 g of dry leaves/200 mL water, twice daily) for six days. CYP1A2, CYP2A6, XO, NAT2 and UGT1A1/1A6 activities were determined before and at the end of the study period, using the following caffeine and paracetamol metabolic ratios: CYP1A2: 17MX/137MX (saliva) and (AFMU+1MU+1MX)/17MU (urine); CYP2A6: 17MU/(17MU + 17MX), XO: 1MU/(1MU+1MX), NAT2, AFMU/(AFMU+1MU+1MX) and UGT1A1/1A6 glucuronidated/total paracetamol, all determined in urine. NAT2 metabolic ratio was significantly reduced following peppermint consumption (0.15 ± 0.13 vs 0.14 ± 0.13; p < 0.05). CYP1A2 urine and saliva indices were reduced, yet not significantly, following peppermint consumption (urine: 3.17 ± 1.08 vs 2.91 ± 0.76, saliva: 0.56 ± 0.12 vs 0.50 ± 0.12; p > 0.05). Peppermint had no influence on CYP2A6, XO and UGT1A1/1A6 indices. Daily ingestion of peppermint tea may alter pharmacokinetics of clinically administered drugs and promote cancer chemoprevention through NAT2 inhibition.

Pyrazoles as potential anti-angiogenesis agents: a contemporary overview.

Angiogenesis is a mulit-step process by which new blood vessels are formed from preexisting vasculature. It is a key rate limiting factor in tumor growth since new blood vessels are necessary to increase tumor size. In this context it has been shown that anti-angiogenic factors can be used in cancer therapy. Among the plethora of heterocyclic compounds administered as anti-angiogenesis agents, pyrazoles constitute one of the bottlenecks of this category. Currently, several pyrazole based compounds are administered or are in Phase II and III trials and new targets emerge. It is highly possible that the advent of the next two decades will lead to the discovery and use of additional pyrazoles whose anti-angiogenic profile will position them in the forefront of the battle of various malignancies. The present review is an attempt to focus on those pyrazoles that arise as anti-angiogenesis agents commenting both on the chemistry and bioactivity that these exhibit aiming to contribute to the perspectives that they hold for future research.

Antiproliferative novel isoxazoles: modeling, virtual screening, synthesis, and bioactivity evaluation.

A series of novel isoxazole derivatives were efficiently synthesized through the adaptation/modification of an in situ synthetic procedure for pyrazoles. All novel compounds were tested against four different cell lines to evaluate their antiproliferative activity. Based on the Hela cells results of this study and previous work, a classification model to predict the anti-proliferative activity of isoxazole and pyrazole derivatives was developed. Random Forest modeling was used in view of the development of an accurate and reliable model that was subsequently validated. A virtual screening study was then proposed for the design of novel active derivatives. Compounds 9 and 11 demonstrated significant cytostatic activity; the fused isoxazole derivative 18 and the virtually proposed compound 2v, were proved at least 10 times more potent as compared to compound 9, with IC50 values near and below 1 µM. In conclusion, a new series of isoxazoles was exploited with some of them exhibiting promising cytostatic activities. Further studies on the substitution pattern of the isoxazole core can potentially provide compounds with cytostatic action at the nM scale. In this direction the in silico approach described herein can also be used to screen existing databases to identify derivatives with anticipated activity.

Resveratrol and related stilbenes: their anti-aging and anti-angiogenic properties.

Dietary stilbenes comprise a class of natural compounds that display significant biological activities of medicinal interest. Among them, their antioxidant, anti-aging and anti-angiogenesic properties are well established and subjects of numerous research endeavors. This mini-review aspires to account and present the literature reports published on research concerning various natural and synthetic stilbenes, such as trans-resveratrol. Special focus was given to most recent research findings, while the mechanisms underlying their anti-aging and anti-angiogenic effects as well as the respective signaling pathways involved were also presented and discussed.

Novel pyrazole and indazole derivatives: synthesis and evaluation of their anti-proliferative and anti-angiogenic activities.

The synthesis of several new pyrazole and indazole derivatives from acetophenone and tetralone substrates is reported. The bioactivities of the new compounds were evaluated through in vitro assays for endothelial cell proliferation and tube formation. Results herein indicate that the easily prepared compounds containing the indazole structural framework exhibit potent cytostatic properties against all cell lines tested, with compounds 13 and 14 being the most active displaying IC(50) values of 1.5 ± 0.4 µM and 5.6 ± 2.5 µM, respectively, against MCF-7 cells. In addition, the indazole derivative 16 was assessed as a competent inhibitor of endothelial tube formation at 30 µM.

Novel pyrazole derivatives: synthesis and evaluation of anti-angiogenic activity.

The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro.

Differential estrogen receptor subtype modulators: assessment of estrogen receptor subtype-binding selectivity and transcription-regulating properties of new cycloalkyl pyrazoles.

Several new cycloalkyl-fused diaryl pyrazoles were synthesized and their binding affinity for the estrogen receptor (ER) subtypes, ERalpha and ERbeta, and subtype-specific agonist/antagonist properties were determined. Cyclopentane- and cyclohexane-fused pyrazoles with p-hydroxyphenyl rings at positions 1 and 3 displayed modest ERbeta-binding selectivity and variable agonism through ERalpha, while behaving as full estrogen antagonists through ERbeta in estrogen-responsive element (ERE)-dependent gene expression assays. By contrast, the 2,3-diphenolic derivatives were non-selective and considerably less effective ERbeta antagonists compared to 1,3-diphenolic ones. The cyclohexane-fused 1,3-diphenolic pyrazole 8, in particular, behaved as full ERalpha agonist/ERbeta antagonist in these assays. Molecular modelling revealed the structural determinants possibly accounting for the differential regulation of transcription through the two ERs exhibited by 8. The data also shows that the ER subtype-binding selectivity and agonist/antagonist efficacy of the 1,3-diphenolic pyrazoles is influenced by the cycloalkyl ring fused to the pyrazole core. Using 8 we show that, though the mutant androgen receptor (AR) of LNCaP cells is required for estrogen as well as androgen stimulation of cell growth, estrogen responsiveness of the cells depends on ERbeta and AR but not on ERalpha.

Headspace solid phase micro extraction gas chromatographic determination of fenthion in human serum.

A simple and effective analytical procedure was developed for the determination of fenthion residues in human serum samples. The sample treatment was performed using the headspace solid-phase micro extraction with polyacrylate fiber, which has the advantage to require low amount of serum (1 mL) without tedious pre-treatment. The quantification of fenthion was carried out by gas chromatography-mass spectrometry and the recoveries ranged from 79 to 104% at two spiking levels for 6 replicates. Detection and quantification limits were calculated as 1.51 and 4.54 ng/mL of serum respectively. Two fenthion metabolites fenoxon and fenthion-sulfoxide were also identified.

Synthesis of novel nitro-substituted triaryl pyrazole derivatives as potential estrogen receptor ligands.

Novel tetrasubstituted pyrazole derivatives bearing a nitro substituent on their A-phenol ring were synthesized and their binding affinity towards the estrogen receptor (ER) subtypes ERalpha and ERbeta was determined. Among compounds tested, the 2-nitrophenol derivative 5c was found to bind satisfactorily to both estrogen receptor subtypes (RBAalpha=5.17 and RBAbeta=3.27). In general, the introduction of a nitro group into the A ring of these compounds was found to benefit their ERbeta binding abilities.

High affinity 17alpha-substituted estradiol derivatives: synthesis and evaluation of estrogen receptor agonist activity.

We synthesized four derivatives of 17beta-estradiol (E2) with an azide substitution on a 17alpha-side chain of varying length, namely 17alpha-(azidopropargyl)-3,17beta-estradiol (5), its 17beta-azido derivative (diazide 7), 17alpha-(5-azido-pent-1-ynyl)-3,17beta-estradiol (6) and 17alpha-(azidopentyn-2-yl)-3,17beta-estradiol (10). While most of the derivatives had low (7) or marginal (6 and 10) relative binding affinity (RBA) for both types of estrogen receptor (ERalpha and ERbeta), the RBAalpha and RBAbeta of 5 were practically identical to those of E2. The estrogenic activity of the derivatives was assessed using estrogen-responsive breast (MCF-7) and endometrial cancer (Ishikawa) cells. While 5 was a potent and effective inducer of alkaline phosphatase in Ishikawa cells and 7 was less potent but as effective as 5, 6 was marginally active and 10 was totally inactive in this respect. In the presence of 0.1 nM E2, however, 6 exhibited some ER antagonist activity at the highest concentration tested (1 microM). Similar results were obtained as regards the potency and efficacy of stimulation of MCF-7 cell proliferation and induction of luciferase gene expression in MCF-7:D5L cells, a clone stably transfected with an estrogen-responsive form of the gene. These data suggest that, while 5, 6, 7 and 10 interact with either type of ER in isolation, only 5 and 7 exhibit substantial ER agonist activity in the different estrogen-target cells examined, which could provide for photoaffinity labelling of the receptor in the cell as well as in isolation.

2-Pyridin-2-yl-1H-indole derivatives: synthesis, estrogen receptor binding affinity, and photophysical properties.

A series of novel 2-pyridin-2-yl-1H-indole derivatives (4a-f) was prepared by intramolecular cyclodehydration of alpha-anilinyl (or 3-anisidyl)-2-pyridin-2-yl-ethanones (2a-f) and their optical spectroscopy and estrogen receptor (ER) binding properties were studied. These compounds showed long wavelength fluorescent emission, which is sensitive to solvent polarity and pH, while indol-6-ols 4b, e, and f displayed reasonably good binding affinities to ER.