Ashwagandha (Withania somnifera) consumption is surging in Europe despite emerging pharmacovigilance reports linking it to hepatotoxicity. Regulatory oversight varies across the continent, ranging from unrestricted sale to strict bans, creating a fragmented landscape. This study investigated how these diverse regulatory frameworks impact online safety disclosures, product labelling compliance, and consumer exposure. We employed a sequential mixed-methods digital surveillance approach across three countries: Hungary (permissive), Poland (restricted limits), and Denmark (banned). First phase of the study involved search engine scraping of 150 websites and content analysis of health claims and safety warnings of 110 relevant identified sellers. Second phase involved test purchases of 12 samples to assess labelling compliance. A "regulatory paradox" was identified across the different jurisdictions. Polish webshops, operating under specific quantitative limits, demonstrated the highest general safety disclosure (n = 37/43; 86.0% included warnings) in Phase I and labelling compliance (n = 24/33; 72.7%) in Phase II. Meanwhile, fully legitimate vendors in Hungary provided limited warnings. In contrast, in Denmark resulted in a polarized market: while 54.84% of webshops (n = 17/31) targeting Danish consumers provided zero safety warnings, an unauthorized "rogue" pharmacy paradoxically provided more detailed clinical contraindication information than legal Hungarian vendors (p < 0.001). Critically, warnings regarding hepatotoxicity were nearly absent on every vendors’ site (n = 2/110). Current fragmented European regulatory framework fails to ensure consumer protection. Poland’s risk-based restriction model combining dosage caps with mandatory labelling appears more effective at protecting consumers than either complete bans or unregulated access, signalling urgent need for a harmonized European regulatory framework.

Carbamazepine (CBZ) is a widely used antiepileptic drug and a persistent aquatic contaminant due to its high global consumption and poor removal by wastewater treatment plants. Although numerous studies have documented its adverse effects on aquatic organisms, knowledge about its impact on learning and the underlying neuronal mechanisms in invertebrates remains limited. Here, we investigated the behavioral and cellular effects of chronic exposure to an environmentally relevant concentration of CBZ (1 μg L-1) in the great pond snail (Lymnaea stagnalis), a well-established model in neuroscience and ecotoxicology. Adult snails were exposed for 21 days, followed by assessments of feeding and locomotion activity, as well as food-reward classical conditioning. According to our results, the exposure did not affect feeding behavior but caused a significant reduction in locomotor activity and a pronounced impairment of associative learning and long-term memory. Locomotor deficits recovered after a 21-day cleaning period, whereas learning impairment persisted, identifying cognition as a particularly vulnerable and long-lasting target of CBZ exposure. Electrophysiological recordings on the Cerebral Giant Cell (CGC), a key serotonergic interneuron involved in food-reward classical conditioning, revealed that CBZ selectively blocked the persistent, but not the transient, component of voltage-gated sodium currents, providing a mechanistic link between CBZ exposure and long-lasting cognitive deficits. These findings demonstrate that CBZ interferes with evolutionarily conserved neuronal targets in invertebrates, yet induces species- and circuit-specific outcomes. From an ecotoxicological perspective, our results pinpoint cognition as a sensitive and persistent endpoint of CBZ pollution and highlight that CBZ may severely compromise individual fitness, with potential consequences for survival and adaptation in natural ecosystems.

Parkinson’s disease (PD) remains a major neurodegenerative disorder with limited therapeutic options. Several in vivo animal studies using different experimental PD models have shown that progesterone (P4) may exert neuroprotective effects; however, the exact cellular and molecular mechanisms, including the receptors mediating these actions of P4, have not been investigated. A recent study from the laboratory of Peter Thomas demonstrated that the selective membrane progesterone receptor α (mPRα/PAQR7) agonist Org OD 02-0 (10-ethenyl-19-norprogesterone) exhibits neuroprotective effects in a human PD cell model, identifying mPRα as a promising target for the development of therapeutic strategies for PD prevention and management. However, the blood-brain barrier (BBB) penetration and in vivo pharmacokinetics of Org OD 02–0 have not yet been examined. Therefore, the aim of the present pilot study was to investigate the BBB penetration and tissue distribution of Org OD 02–0 following systemic administration in rats. To achieve this, we first developed a sensitive high-performance liquid chromatography-coupled mass spectrometry method for the detection and quantification of Org OD 02–0 in rat tissues. Using this method, we investigated the distribution of Org OD 02–0 in male Wistar rats following subcutaneous administration (2.6 mg/kg). Org OD 02–0 was detectable in all examined tissues, showing higher concentrations in the brain (frontal lobe) and kidney and lower levels in plasma and liver at 1, 3, 6, and 12 h post-injection. The concentration data suggested differential tissue distribution and potential preferential accumulation in the kidneys. Importantly, our results demonstrate, for the first time, that Org OD 02–0 crosses the BBB and reaches pharmacologically relevant concentrations in the central nervous system following systemic administration, supporting its potential as a candidate for PD therapy. Based on these data, in vivo studies in rat PD models are warranted to further evaluate the therapeutic efficacy of Org OD 02-0.

Over the last 20 years, tributyltin (TBT) has been reported to cause metabolic disruption in both invertebrates and vertebrates, highlighting the need for further detailed analysis of its physiological effects. This study aimed to investigate the metabolic-disrupting effects of TBT from the behavioral to the molecular level. Adult specimens of the great pond snail (Lymnaea stagnalis) were exposed to an environmentally relevant concentration (100 ng L-1) of TBT for 21 days. After the chronic exposure, behavioral alterations as well as histological, cellular, and molecular changes were investigated in the central nervous system, kidney, and hepatopancreas. TBT exposure significantly decreased feeding activity, while locomotor activity remained unchanged. At the histological level, the cellular localization of tin was demonstrated in all tissues investigated and, in addition, characteristic morphological changes were observed in the kidney and hepatopancreas. Tissue-specific changes in lipid profiles confirmed TBT-induced disruption of lipid homeostasis in mollusks, characterized by a consistent reduction in the proportion of polyunsaturated fatty acids and a shift toward more saturated lipids. The expression of 17β-hydroxysteroid dehydrogenase type 12 (HSD17B12) enzyme, involved in lipid metabolism in vertebrates, was reduced in all three tissues after TBT exposure. Our results show that TBT induces significant multi-level metabolic changes in Lymnaea, including direct alterations in feeding activity and lipid composition. Our findings also suggest that HSD17B12 enzyme plays a key role in lipid metabolism in mollusks, as in mammals, and is likely involved in TBT-induced metabolic disruption. Overall, our study extends the findings of previous studies on mollusks by providing novel behavioral as well as tissue-specific histological and metabolic data and highlights the complexity and evolutionary conserved way of TBT-induced metabolic disruption.

The joint risks assessment of thermal stress and rising loads of pharmaceuticals (PhACs) in surface waters is a relevant topic in aquatic ecotoxicology. This study investigated the relevance of increased water temperature to alter the acute toxicity of environmentally relevant carbamazepine (CBZ), citalopram (CIT) and tramadol (TRA) concentrations as mixtures (ECs) and delayed outcomes in Daphnia magna. Responses of detoxification and antioxidant pathways in premature daphnids post an acute 24 h (pulsed) exposure to the PhACs mixtures and delayed responses as the reproductive output over 14 days recovery were investigated under 21- and 26 °C incubation. Biphasic modulation in glutathione S-transferase (GST) activity and significant inhibition of superoxide dismutase (SOD) activity were observed in both thermal regimes with significant shift in effective thresholds from 10-fold ECs at 21 °C to ECs at 26 °C incubation. Significant induction in catalase (CAT) activity and oxidative stress development were recorded at elevated temperatures from the 10-fold ECs dose onward. Pulsed exposures at 26 °C also led to significant decrease in the reproduction of daphnids above the 10-fold ECs of PhACs. The Integrated Biomarker Response scoring (IBRv2) approach outlined a 1.8-fold increase in alterations of daphnids exposed to 100-fold ECs of PhACs at 26 °C.

MicroRNAs (miRNAs) are key regulators of gene expression, shaping neuroplasticity, stress responses, and neuromodulation. In the pond snail Lymnaea stagnalis, inhibition of the miRNA-processing enzyme Dicer with Poly-l-Lysine (PLL) has been shown to impair long-term memory (LTM) formation, yet the molecular pathways affected remain unclear. Here, we examined PLL injection's transcriptional and cellular/neurochemical effects in untrained (i.e., non-associatively conditioned) snails. We focused on genes involved in neuroplasticity (LymGRIN1, LymCREB1), stress response (LymHSP70), and serotonergic/dopaminergic signaling (LymTPH, LymSERT, LymDDC), along with measurements of the levels of serotonin, dopamine, and HSP70 protein. We found that PLL did not alter the expression of memory-related genes in the non-associatively conditioned snails. However, we observed a marked downregulation of LymTPH and LymSERT, which was accompanied by a significant reduction in the level of serotonin. In contrast, the expression of LymDDC and the level of dopamine remained unchanged. Although we detected a significant upregulation in the expression of LymHSP70, indicating a strong stress response, the level of HSP70 protein did not change significantly. These findings suggest that PLL-induced Dicer inhibition primarily disrupts serotonin homeostasis, potentially altering motivation and feeding behaviour rather than directly impairing memory processes. This study highlights the broader role of miRNA pathways in regulating stress resilience and neuromodulation. By linking miRNA activity to serotonergic signaling, our findings suggest that miRNAs influence behavioural states beyond synaptic plasticity, with potential implications for understanding how miRNA dysregulation affects mood, motivation, and cognitive function across species.

Organic UV-filters in surface waters have gained increasing attention, yet their analytical detection and quantification remain challenging. In this study, we developed an analytical method involving optimized solid phase extraction (SPE) and UHPLC-MS/MS to simultaneously analyze selected top UV-filter compounds commonly used in Europe and/or listed in the 5th Watch List (2025) under the Water Framework Directive: avobenzone (AVO), iscotrizinol (DBT), octinoxate (EHMC), ethylhexyl triazone (EHT), and octocrylene (OC). The validation criteria included the evaluation of selectivity, linearity, accuracy, LOD, and LOQ. Plackett-Burman design as a comprehensive and high-level approach was applied to evaluate robustness. Among the UHPLC-MS/MS variables tested, the most influential factors were the mobile phase pH, oven temperature, and mobile phase flow rate. Instead of using the commonly applied C18 and C8 HPLC columns, a pentafluorophenyl column was used to achieve a better and optimal chromatographic separation. The validated method achieved accuracies from 85.9 % to 123.4 % and LOD/LOQ values from 0.76/2.29 ng/L to 3.16/9.58 ng/L. With the optimized SPE protocol using Phenomenex Si-1 cartridges, the recoveries ranged from 45.2 % to 73.4 %. The method was applied for the analysis of Lake Balaton's surface freshwater, detecting all targeted UV-filters at the following concentrations: c (AVO) = 590.5 f 17.5 ng/L, c(DBT) = 136 f 9.7 ng/L, c(EHMC) = 776.8 f 19.2 ng/L; c(EHT) = 55.2 f 4.2 ng/ L, and c(OC) = 141.8 f 7.2 ng/L. Our study provides the first analytical protocol for DBT and EHT measurements. This study reflects the continuously evolving nature of UV-filters in sunscreens, emphasizing the need for new and adaptive analytical approaches. Our method innovatively evaluates and optimizes multiple parameters, ensuring robust and reliable quantification of five top organic UV-filters in environmental samples.

Ecdysone-controlled pathways regulate ecdysozoan development through both a nuclear (nEcR) and a membrane receptor (mEcR). Ecdysone signaling was long thought to be exclusive to ecdysozoans; however, studies since 2010 have demonstrated the presence of an orthologue of the nEcR/NR1H receptor in other protostome taxa, including mollusks and annelids. Nevertheless, there is still no clear consensus regarding the presence or functional role of ecdysone-like signaling in mollusks. This study aimed to advance our understanding of the evolution of ecdysone signaling by investigating the potential synthesis and physiological role of 20-hydroxyecdysone (20HE) in the great pond snail ( Lymnaea stagnalis ) by applying a complex experimental approach. Exposure to different concentrations (10 ng/L, 100 ng/L, and 1 µg/L) of 20HE had no effect on hatching, heart rate, locomotion (gliding), or feeding in Lymnaea embryos. Furthermore, the treatments did not alter the expression of Lymnaea homologs of Drosophila nEcR and mEcR. Using cluster analysis and phylogenetics to resolve the evolution of Halloween genes involved in ecdysone synthesis, we found no clear homologues of Spook, Disembodied, or Shadow in any lophotrochozoan species, while Phantom and Shade appear to have a many to many homologues in lophotrochozoans. Although mass spectrometric analysis detected trace concentrations of 20HE in various tissues of adult Lymnaea specimens, we suggest that its presence is of dietary origin. Our findings clearly indicate that an endogenous ecdysone synthesis pathway is not present in Lymnaea and that 20HE has no effect on the physiology of Lymnaea embryos. Although further experiments are required on other molluscan models as well, we propose that molluscan sequences homologous to Drosophila nEcR and mEcR are unlikely to function as ecdysone receptors, and that ecdysone-like signaling is absent in lophotrochozoans.

BackgroundOver the past 4 decades, obesity has escalated into a global epidemic, with its worldwide prevalence nearly tripling. Pharmacological treatments have evolved with the recent development of glucagon-like peptide 1 agonists, such as semaglutide. However, off-label use of drugs such as Ozempic for cosmetic weight loss has surged in popularity, raising concerns about potential misuse and the emergence of substandard and falsified products in the unregulated supply chain. ObjectiveThis study aims to conduct a multifactor investigation of product quality and patient safety risks associated with the unregulated online sale of semaglutide by examining product availability and vendor characteristics and assessing product quality through test purchases. MethodsWe used a complex risk and quality assessment methodology combining online market surveillance, search engine results page analysis, website content assessment, domain traffic analytics, conducting targeted product test purchases, visual quality inspection of product packaging, microbiological sterility and endotoxin contamination evaluation, and quantitative sample analysis using liquid chromatography coupled with mass spectrometry. ResultsWe collected and evaluated 1080 links from search engine results pages and identified 317 (29.35%) links belonging to online pharmacies, of which 183 (57.7%) led to legal pharmacies and 134 (42.3%) directed users to 59 unique illegal online pharmacy websites. Web traffic data for the period between July and September 2023 revealed that the top 30 domains directly or indirectly affiliated with illegal online pharmacies accumulated over 4.7 million visits. Test purchases were completed from 6 illegal online pharmacies with the highest number of links offering semaglutide products for sale without prescription at the lowest price range. Three injection vial purchases were delivered; none of the 3 Ozempic prefilled injection pens were received due to nondelivery e-commerce scams. All purchased vials were considered probable substandard and falsified products, as visual inspection indicated noncompliance in more than half (59%-63%) of the evaluated criteria. The semaglutide content of samples substantially exceeded labeled amounts by 28.56%-38.69%, although no peptide-like impurities were identified. The lyophilized peptide samples were devoid of viable microorganisms at the time of testing; however, endotoxin was detected in all samples with levels ranging between 2.1645 EU/mg and 8.9511 EU/mg. Furthermore, the measured semaglutide purity was significantly low, ranging between 7.7% and 14.37% and deviating from the 99% claimed on product labels by manufacturers. ConclusionsGlucagon-like peptide 1 agonist drugs promoted for weight loss, similar to erectile dysfunction medications more than 2 decades ago, are becoming the new blockbuster lifestyle medications for the illegal online pharmacy market. Protecting the pharmaceutical supply chain from substandard and falsified weight loss products and raising awareness regarding online medication safety must be a public health priority for regulators and technology platforms alike.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

This qualitative study assesses the quality, amount of active ingredient, and characteristics associated with counterfeiting of semaglutide purchased from illegal online pharmacies without a prescription.

In this study, the phytochemical composition, in vitro antioxidant, and anti-inflammatory effects of the aqueous and 60% ethanolic (EtOH) extracts of Santolina rosmarinifolia leaf, flower, and root were examined. The antioxidant activity of S. rosmarinifolia extracts was determined by 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. The total phenolic content (TPC) of the extracts was measured by the Folin–Ciocalteu assay. The anti-inflammatory effect of the extracts was monitored by the Griess assay. The chemical composition of S. rosmarinifolia extracts was analysed using the LC-MS technique. According to our findings, 60% EtOH leaf extracts showed the highest Trolox equivalent antioxidant capacity (TEAC) values in both ABTS (8.39 ± 0.43 µM) and DPPH (6.71 ± 0.03 µM) antioxidant activity assays. The TPC values of the samples were in good correspondence with the antioxidant activity measurements and showed the highest gallic acid equivalent value (130.17 ± 0.01 µg/mL) in 60% EtOH leaf extracts. In addition, the 60% EtOH extracts of the leaves were revealed to possess the highest anti-inflammatory effect. The LC-MS analysis of S. rosmarinifolia extracts proved the presence of ascorbic acid, catalpol, chrysin, epigallocatechin, geraniol, isoquercitrin, and theanine, among others, for the first time. However, additional studies are needed to investigate the direct relationship between the chemical composition and physiological effects of the herb. The 60% EtOH extracts of S. rosmarinifolia leaves are potential new sources of natural antioxidants and anti-inflammatory molecules in the production of novel nutraceutical products.

Infertility affects millions worldwide, posing a significant global health challenge. The proteomic analysis of follicular fluid provides a comprehensive view of the complex molecular landscape within ovarian follicles, offering valuable information on the factors influencing oocyte development and on the overall reproductive health. The follicular fluid is derived from the plasma and contains various proteins that can have different roles in oocyte health and infertility, and this fluid is a critical microenvironment for the developing oocytes as well. Using the high-performance liquid chromatography-mass spectrometry method, we investigated the protein composition of the follicular fluid, and after classification, we carried out relative quantification of the identified proteins in the pregnant (P) and non-pregnant (NP) groups. Based on the protein–protein interaction analysis, albumin and apolipoprotein A1 (ApoA1) were found to be hub proteins, and the quantitative comparison of the P and NP groups resulted in a significantly lower concentration of ApoA1 and high-density lipoprotein cholesterol in the P group. As both molecules are involved in the cholesterol transport, we also investigated their role in the development of oocytes and in the prediction of fertility.

The presence of microplastics (MPs) in the global ecosystem has generated a rapidly growing concern worldwide. Although their presence in the marine environment has been well-studied, much less data are available on their abundance in freshwaters. MPs alone and in combination with different chemicals has been shown to cause acute and chronic effects on algae and aquatic invertebrate and vertebrate species at different biological levels. However, the combined ecotoxicological effects of MPs with different chemicals on aquatic organisms are still understudied in many species and the reported data are often controversial. In the present study, we investigated, for the first time, the presence of MPs in Lake Balaton, which is the largest shallow lake of Central Europe and an important summer holiday destination. Moreover, we exposed neonates of the well-established ecotoxicological model organism Daphnia magna to different MPs (polystyrene [3 μm] or polyethylene [≤ 100 μm]) alone and in combination with three progestogen compounds (progesterone, drospirenone, levonorgestrel) at an environmentally relevant concentration (10 ng L-1) for 21 days. The presence of 7 polymer types of MPs in the size range of 50-100 μm was detected in Lake Balaton. Similarly to the global trends, polypropylene and polyethylene MPs were the most common types of polymer. The calculated polymer-independent average particle number was 5.5 particles m-3 (size range: 50 μm - 100 μm) which represents the values detected in other European lakes. Our ecotoxicological experiments confirmed that MPs and progestogens can affect D. magna at the behavioral (body size and reproduction) and biochemical (detoxification-related enzyme activity) levels. The joint effects were negligible. The presence of MPs may lead to reduced fitness in the aquatic biota in freshwaters such as Lake Balaton, however, the potential threat of MPs as vectors for progestogens may be limited.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17β-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

The neuroprotective effects of environmental enrichment and PACAP (pituitary adenylate cyclase-activating polypeptide) are well-described in Parkinson's disease. The aim of our study is to investigate the beneficial effects of these factors in aging parkinsonian rats. Newborn Wistar rats were divided into standard and enriched groups according to their environmental conditions. Standard animals were raised under regular conditions. During the first five postnatal weeks, enriched pups were placed in larger cages with different objects. Aging animals received (1) saline, (2) 6-hydroxidopamine (6-OHDA), or (3) 6-OHDA + PACAP injections into the left substantia nigra (s.n.). On the seventh postoperative day, the left and right s.n. were collected. The s.n. of young and aging unoperated animals were also examined in our experiment. We determined the dopamine (DA) levels by the HPLC-MS technique, while the sandwich ELISA method was used to measure the Parkinson disease protein 7 (PARK7) protein levels. In healthy animals, we found an age-related decrease of DA levels. In aging parkinsonian-enriched rats, the operation did not result in a significant DA loss. PACAP treatment could prevent the DA loss in both the standard and enriched groups. All injured PACAP-treated rats showed remarkably higher protective PARK7 levels. The protective effect of PACAP correlated with the increase of the DA and PARK7 levels.

Grape pomace is a valuable source of various bioactive compounds such as plant-derived polyphenols and polyunsaturated fatty acids (PUFAs). The commercial demand of grape skin and seed powders as nutraceuticals is still growing. However, no distinction is currently made between unfermented native grape seed and grape seed pomace powders regarding their antioxidant activities. Our aim was to find the relationship between the polyphenol and fatty acid content as well as the antioxidant capacity of native and fermented grape seeds of four different grape varieties harvested in the Villány wine region. According to our results, none of the three investigated polyphenols (resveratrol, rutin, quercetin) could be detected in native grape seed samples in correlation with their significantly lower total antioxidant capacities compared to fermented seed samples. Pinot Noir (PN) grape seed pomace samples with the highest resveratrol and oil content showed significantly higher total antioxidant capacity than Cabernet Sauvignon (CS), Syrah (S) and Blue Portugal (BP) samples. Based on the statistical analysis, positive correlation was found between the fatty acid content and the resveratrol concentration in the pomace samples of different grape varieties. In contrast, rutin concentrations were negatively proportional to the fatty acid content of the fermented samples. No significant correlation was found considering the quercetin content of the samples. According to our findings, grape pomace seems a more promising source in the production of nutraceuticals, since it contains polyphenols in higher concentration and exerts significantly higher antioxidant activity than native grape seeds.

The presence of sex steroid hormones in aquatic ecosystems is of rapidly growing concern worldwide since they can affect the different non-target species including cladocerans. Although data are available on the effects of estrogens on the well-established ecotoxicological model organism Daphnia magna, the molecular or behavioural alterations induced by environmentally relevant concentrations (from a few ng L−1 to a few hundred ng L−1 in average) of progestogens have not been investigated on this species. In the present study, we exposed neonates of D. magna to relevant equi-concentrations (1, 10, 100, 500 ng L−1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) in short-term (6 days) and long-term (21 days) experiments. Significant alterations were observed at the molecular, cellular, and individual levels. During the short-term exposure, all of the mixtures increased the gene expression of glutathione S-transferase (GST) detoxification enzyme, moreover, the activity of GST was also significantly increased at the concentrations of 10, 100, and 500 ng L−1. In long-term exposure, the number of days until production of the first eggs was reduced at the 10 ng L−1 concentration compared to control, furthermore, the maximum egg number per individual increased at the concentrations of 1 and 10 ng L−1. Based on the authors' best knowledge, this is the first study to investigate the effects of progestogens in mixtures and at environmentally relevant concentrations on D. magna. Our findings contribute to the understanding of the possible physiological effects of human progestogens. Future research should be aimed at understanding the potential mechanisms (e.g., perception) underlying the changes induced by progestogens.

Perception and processing of chemical cues are crucial for aquatic gastropods, for proper elaboration of adaptive behavior. The pond snail, Lymnaea stagnalis, is a model species of invertebrate neurobiology, in which peripheral sensory neurons with different morphology and transmitter content have partly been described, but we have little knowledge regarding their functional morphological organization, including their possible peripheral intercellular connections and networks. Therefore the aim of our study was to characterize the sensory system of the tentacles and the lip, as primary sensory regions, and the anterior foot of Lymnaea with special attention to the transmitter content of the sensory neurons, and their relationship to extrinsic elements of the central nervous system. Numerous bipolar sensory cells were demonstrated in the epithelial layer of the peripheral organs, displaying immunoreactivity to antibodies raised against tyrosine hydroxylase, histamine, glutamate and two molluscan type oligopeptides, FMRFamide and Mytilus inhibitory peptide. A subepithelial plexus was formed by extrinsic serotonin and FMRFamide immunoreactive fibers, whereas in deeper regions axon processess of different origin with various immunoreactivities formed networks, too. HPLC–MS assay confirmed the presence of the low molecular weight signal molecules in the three examined areas. Following double-labeling immunohistochemistry, close arrangements were observed, formed by sensory neurons and extrinsic serotonergic (and FMRFamidergic) fibers at axo-dendritic, axo-somatic and axo-axonic levels. Our results suggest the involvement of a much wider repertoire of signal molecules in peripheral sensory processes of Lymnaea, which can locally be modified by central input, hence influencing directly the responses to environmental cues.