Pharmaceuticals in water and sediment of small streams under the pressure of urbanization: Concentrations, interactions, and risks.

Small streams are crucial but vulnerable elements of ecological networks. To better understand the occurrence of pharmaceutically active compounds (PhACs) in streams, this study focused on the occurrence, distribution, and environmental risk of 111 PhACs and 7 trace elements based on a total of 141 water and sediment samples from small streams located in the urbanizing region of Budapest, Hungary. Eighty-one PhACs were detected in the aqueous phase, whereas sixty-two compounds were detected in the sediment. Carbamazepine (CBZ) was the most frequently identified PhAC in water, and was found in 91.5% of all samples. However, the highest concentrations were measured for lamotrigine (344.8 µg·L-1) and caffeine (221.4 µg·L-1). Lidocaine was the most frequently occurring PhAC in sediment (73.8%), but the maximum concentrations were detected for CBZ (395.9 ng·g-1) and tiapride (187.7 ng·g-1). In both water and sediment, more PhACs were found downstream of the wastewater treatment plants (WWTPs) than in the samples not affected by treated wastewater, even though no relationship was observed between the total amount of treated wastewater and the number of detected PhACs. The PhAC concentrations were also independent of the distance from the WWTP effluents. PhAC-polluted samples were detected upstream of the WWTPs, thereby suggesting the relevance of diffuse emissions in addition to WWTP outlets. The most frequently detected PhACs in the sediment were usually also present in the water samples collected at the same place and time. The varying concentrations of PhACs and the fluctuating water-sediment properties resulted in a lack of correlation between the general chemical properties and the concentrations of PhACs, which makes it difficult to predict PhAC contamination and risks in urbanized small streams. The environmental risk assessment indicated that diclofenac had the highest risk in the sampling area.

Neuropeptide Localization in Lymnaea stagnalis: From the Central Nervous System to Subcellular Compartments.

Due to the relatively small number of neurons (few tens of thousands), the well-established multipurpose model organism Lymnaea stagnalis, great pond snail, has been extensively used to study the functioning of the nervous system. Unlike the more complex brains of higher organisms, L. stagnalis has a relatively simple central nervous system (CNS) with well-defined circuits (e.g., feeding, locomotion, learning, and memory) and identified individual neurons (e.g., cerebral giant cell, CGC), which generate behavioral patterns. Accumulating information from electrophysiological experiments maps the network of neuronal connections and the neuronal circuits responsible for basic life functions. Chemical signaling between synaptic-coupled neurons is underpinned by neurotransmitters and neuropeptides. This review looks at the rapidly expanding contributions of mass spectrometry (MS) to neuropeptide discovery and identification at different granularity of CNS organization. Abundances and distributions of neuropeptides in the whole CNS, eleven interconnected ganglia, neuronal clusters, single neurons, and subcellular compartments are captured by MS imaging and single cell analysis techniques. Combining neuropeptide expression and electrophysiological data, and aided by genomic and transcriptomic information, the molecular basis of CNS-controlled biological functions is increasingly revealed.

Effects of pharmaceutically active compounds (PhACs) on fish body and scale shape in natural waters.

BACKGROUND: In recent years, there are growing concerns about pharmaceutically active compounds (PhACs) in natural ecosystems. These compounds have been found in natural waters and in fish tissues worldwide. Regarding their growing distribution and abundance, it is becoming clear that traditionally used risk assessment methodologies and ecotoxicological studies have limitations in several respects. In our study a new, combined approach of environmental impact assesment of PhACs has been used. METHODS: In this study, the constant watercourses of the suburban region of the Hungarian capital (Budapest) were sampled, and the body shape and scale shape of three fish species (roach Rutilus rutilus, chub Squalius cephalus, gibel carp Carassius gibelio) found in these waters were analyzed, based on landmark-based geometric morphometric methods. Possible connections were made between the differences in body shape and scale shape, and abiotic environmental variables (local- and landscape-scale) and measured PhACs. RESULTS: Significant connections were found between shape and PhACs concentrations in several cases. Despite the relatively large number of compounds (54) detected, citalopram, propranolol, codeine and trimetazidine significantly affected only fish body and scale shape, based on their concentrations. These four PhACs were shown to be high (citalopram), medium (propranolol and codeine), and low (trimetazidine) risk levels during the environmental risk assessment, which were based on Risk Quotient calculation. Furthermore, seven PhACs (diclofenac, Estrone (E1), tramadol, caffeine 17α-Ethinylestradiol (EE2), 17α-Estradiol (aE2), Estriol (E3)) were also categorized with a high risk level. However, our morphological studies indicated that only citalopram was found to affect fish phenotype amongst the PhACs posing high risk. Therefore, our results revealed that the output of (traditional) environmental/ecological risk assessment based on ecotoxicological data of different aquatic organisms not necessarily show consistency with a "real-life" situation; furthermore, the morphological investigations may also be a good sub-lethal endpoint in ecotoxicological assessments.

Illicit Drugs as a Potential Risk to the Aquatic Environment of a Large Freshwater Lake after a Major Music Festival.

The present study strengthens the view that residues of drugs of abuse may become widespread surface water contaminants following a local music festival. Overall, 10 illicit drugs were detected from the aquatic environment after the festival; cocaine and 3,4-methylenedioxymethamphetamine were present in the highest concentrations. The presence of illicit drugs and their metabolites over 3 monitored festival yr suggested that consumption of these drugs was temporally linked with events. Weather conditions seriously influenced detection of contaminants deriving from events at the lakeshore. Most of the illicit drugs retained their pharmacological activities, with a potentially adverse impact on wildlife. Environ Toxicol Chem 2021;40:1491-1498. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Alterations of Nigral Dopamine Levels in Parkinson's Disease after Environmental Enrichment and PACAP Treatment in Aging Rats.

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.

Environmental risk assessment of pharmaceuticals at a seasonal holiday destination in the largest freshwater shallow lake in Central Europe.

The presence of pharmacologically active compounds (PhACs) in surface waters poses an environmental risk of chronic exposure to nontarget organisms, which is a well-established and serious concern worldwide. Our aim was to determine the temporal changes in ecological risk quotient (RQ) based on the concentrations of 42 PhACs from six sampling sites on seven sampling dates in the water of a freshwater lake in Central Europe preferentially visited by tourists. Our hypothesis was that the environmental risk increases during the summer holiday season due to the influence of tourists. Different experimental toxicological threshold concentrations and seasonal measured environmental concentrations of 16 PhACs were applied to ecological risk assessment. RQs of 4 dominant PhACs (diclofenac, estrone [E1], estradiol [E2], and caffeine) indicated high ecological risk (RQ > 1) for freshwater ecosystems. Additionally, our results confirmed the assumptions that the high tourist season had a significant impact on the calculated RQ; however, these results are mainly due to the concentration and temporal change of particular PhACs, including diclofenac (5.3-419.4 ng/L), E1 (0.1-5.5 ng/L), and E2 (0.1-19.6 ng/L). The seasonal dependent highest RQs changed as follows: 9.80 (June 2017; E2), 1.23 (August 2017; E1), 0.43 (November 2017; E1), 0.51 (April 2018; E1), 5.58 (June 2018, diclofenac), 39.50 (August 2018; diclofenac), and 30.60 (October 2018; diclofenac).

Chemical-neuroanatomical organization of peripheral sensory-efferent systems in the pond snail (Lymnaea stagnalis).

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.

Dataset of pharmaceuticals in the Danube and related drinking water wells in the Budapest region.

The present dataset provides data on the pharmaceutically active compounds (PhACs) concentrations measured in the Danube and the drinking water abstraction wells (DWAW) in the Budapest region. Grab samples were collected during five periods. One hundred and seven water samples from the Danube and ninety water samples from the relevant DWAWs were analyzed to quantify physical-chemical parameters, trace element concentrations, and one hundred and eleven PhACs, including pharmaceutical derivatives, illicit drugs, and alkaloids. The ion concentrations were measured using dual channel ion chromatography, spectrophotometric and titrimetric methods, and inductively coupled plasma mass spectrometry. PhACs concentrations were measured after solid-phase extraction applying supercritical fluid chromatography coupled with tandem mass spectrometry. Fifty-two PhACs were quantified in the Danube, and ten PhACs were present in >80% of the samples. Whereas thirty-two PhACs were quantified in the DWAWs. The present dataset is useful for further comparisons and meta-analyses.

Testing the Applicability of MALDI-TOF MS as an Alternative Stock Identification Method in a Cryptic Species Complex.

Knowledge of intraspecific variability of a certain species is essential for their long-term survival and for the development of conservation plans. Nowadays, molecular/genetic methods are the most frequently used for this purpose. Although, the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has become a promising alternative tool to specify intraspecific variability, there is a lack of information about the limitations of this method, and some methodological issues need to be resolved. Towards this goal, we tested the sensitivity of this method on an intraspecific level, using genetically identified individuals of a cryptic fish species complex collected from five distinct populations. Additionally, some methodologic issues, such as the effect of (1) delayed sample preparation, (2) clove oil anaesthetization, and (3) different tissue types (muscle, and brain) were investigated using the MS analysis results. Our results show that the delayed sample preparation has a fundamental effect on the result of MS analysis, while at the same time the clove oil did not affect the results considerably. Both the brain and muscle samples were usable for cryptic species identification, but in our opinion this method has limited applicability for population-level segregation. The application of MALDI-TOF MS to the exploitable toolkit of phylogenetic and taxonomic researches could be used to broaden conclusions.

Occurrence of pharmaceuticals in the Danube and drinking water wells: Efficiency of riverbank filtration.

Surface waters are becoming increasingly contaminated by pharmaceutically active compounds (PhACs), which is a potential risk factor for drinking water quality owing to incomplete riverbank filtration. This study examined the efficiency of riverbank filtration with regard to 111 PhACs in a highly urbanized section of the river Danube. One hundred seven samples from the Danube were compared to 90 water samples from relevant drinking water abstraction wells (DWAW) during five sampling periods. The presence of 52 PhACs was detected in the Danube, the quantification of 19 agents in this section of the river was without any precedent, and 10 PhACs were present in >80% of the samples. The most frequent PhACs showed higher concentrations in winter than in summer. In the DWAWs, 32 PhACs were quantified. For the majority of PhACs, the bank filtration efficiency was >95%, and not influenced by concentrations measured in the river. For carbamazepine lidocaine, tramadol, and lamotrigine, low (<50%) filtration efficiency was observed; however, no correlations were observed between the concentrations detected in the Danube and in the wells. These frequently occurring PhACs in surface waters have a relatively even distribution, and their sporadic appearance in wells is a function of both space and time, which may be caused by the constantly changing environment and micro-biological parameters, the dynamic operating schedule of abstraction wells, and the resulting sudden changes in flow rates. Due to the changes in the efficiency of riverbank filtration in space and time, predicting the occurrence and concentrations of these four PhACs poses a further challenge to ensuring a safe drinking water supply.

Thermal baths as sources of pharmaceutical and illicit drug contamination.

Despite the fact that there are tens of thousands of thermal baths in existence, knowledge about the occurrence of pharmaceutically active compounds (PhACs) in untreated thermal wastewater is very limited. Because used thermal water is typically legally discharged into surface waters without any treatment, the effluent poses environmental risks for the receiving water bodies. The aim of this study was to show the occurrence patterns and spatiotemporal characteristics of 111 PhACs in thermal wastewater. Six thermal water outflows of different thermal baths were tested in different seasons in the Budapest metropolitan region (Hungary), and diurnal analysis was performed. After solid-phase extraction, the samples were analysed and quantified by coupling supercritical fluid chromatography and mass spectrometry to perform simultaneous multi-residue drug analysis. The results confirm that water discharge pipes directly transport pharmaceuticals into surface water bodies; 34 PhACs were measured to be over the limit of quantification at least once, and 21 of them were found in more than one water sample. The local anaesthetic drug lidocaine, antiepileptic carbamazepine, analgesic derivative tramadol and illicit drug cocaine were detected in more than half of the samples. Caffeine, metoprolol and bisoprolol (cardiovascular drugs), benzoylecgonine (cocaine metabolite), diclofenac (NSAID), citalopram (antidepressant) and certain types of hormones also have a significant frequency of 30-50%. However, the occurrence and concentrations of PhACs vary according to the season and number/types of visitors. As demonstrated by the diurnal fluctuation, drug contamination of thermal waters can significantly vary, even for similar types of baths; furthermore, the quantity and types of some pollutants rapidly change in the discharged thermal wastewater.

Spatiotemporal variations of pharmacologically active compounds in surface waters of a summer holiday destination.

The release of pharmacologically active compounds (PhACs) into aquatic ecosystems poses an environmental risk resulting in a chronic exposure of non-target organisms. A great variety of PhACs, of generally low concentrations, and the complicated sample preparation, makes circumstantial the accurate detection and quantification. Additionally, there is little information published about the spatiotemporal variation of the PhAC load in a larger catchment area utilised for touristic purposes. In addition to the natural biotic and abiotic changes, the seasonal variation of tourism also has a dramatic impact on water quality and the natural ecosystem in larger catchment areas. Therefore, our aim was to develop a reliable solid-phase extraction (SPE)-supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) method for simultaneous multi-residue analysis of drugs to reveal the spatiotemporal changes in the PhAC contaminations in the waters of an important touristic region, the catchment area of the largest shallow lake in Central Europe, Lake Balaton (Hungary). The environmental application of the developed method revealed 69 out of the traced 134 chemical compounds, including 15 PhACs, which were detected from natural waters for the first time. Wastewater treatment plant (WWTP) loads have a major role in the PhAC contamination of the studied area; at the same time, the mass tourism-induced PhAC contamination was also detectable. Furthermore, the impact of tourism was indicated by elevated concentrations of recreational substances (e.g., caffeine and illicit drugs) in the touristic season affecting the water quality of this important summer holiday destination.

Serotonergic regulation of the buccal (feeding) rhythm of the pond snail, Lymnaea stagnalis. An immunocytochemical, biochemical and pharmacological approach.

Hatching is an important phase of the development of pulmonate gastropods followed by the adult-like extracapsular foraging life. Right before hatching the juveniles start to display a rhythmic radula movement, executed by the buccal complex, consisting of the buccal musculature (mass) and a pair of the buccal ganglia. In order to have a detailed insight into this process, we investigated the serotonergic regulation of the buccal (feeding) rhythm in 100% stage embryos of the pond snail, Lymnaea stagnalis, applying quantitative immunohistochemistry combined with the pharmacological manipulation of the serotonin (5-HT) synthesis, by either stimulating (by the 5-HT precursor 5-hydroxytryptophan, 5-HTP) or inhibiting (by the 5-HT synthesis blocker para-chlorophenylalanine, pCPA) it. Corresponding to the direction of the drug effect, significant changes of the fluorescence intensity could be detected both in the cerebral ganglia and the buccal complex. HPLC-MS assay demonstrated that 5-HTP increased meanwhile pCPA decreased the 5-HT content both of the central ganglia and the buccal complex. As to the feeding activity, 5-HTP induced only a slight (20%) increase, whereas the pCPA resulted in a 20% decrease of the radula protrusion frequency. Inhibition of 5-HT re-uptake by clomipramine reduced the frequency by 75%. The results prove the role of both central and peripheral 5-HTergic processes in the regulation of feeding activity. Application of specific receptor agonists and antagonists revealed that activation of a 5-HT1-like receptor depressed the feeding activity, meanwhile activation of a 5-HT6,7-like receptor enhanced it. Saturation binding plot of [3H]-5-HT to receptor and binding experiments performed on membrane pellets prepared from the buccal mass indicated the presence of a 5-HT6-like receptor positively coupled to cAMP. The results suggest that 5-HT influences the buccal (feeding) rhythmic activity in two ways: an inhibitory action is probably exerted via 5-HT1-like receptors, while an excitatory action is realized through 5-HT6,7-like receptors.

Investigation of estrogen activity in the raw and treated waters of riverbank infiltration using a yeast estrogen screen and chemical analysis.

Exposure to various endocrine disrupting chemicals (EDCs) can lead to adverse effects on reproductive physiology and behavior in both animals and humans. An adequate strategy for the prevention of environmental contamination and eliminating the effects of them must be established. Chemicals with estrogenic activity were selected, and the effectiveness of their removal during the purification processes in two drinking water treatment plants (DWTPs) using riverbank infiltrated water was determined. Thirty-five water samples in two sampling campaigns throughout different seasons were collected and screened with a yeast estrogen test; furthermore, bisphenol A (BPA), 17ß-estradiol (E2) and ethinyl-estradiol (EE2) content were measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Our results confirm that estrogenic compounds are present in sewage effluents and raw surface river water of DWTPs. Very low estrogen activity and pg/L concentrations of BPA and E2 were detected during drinking water processing and occasionally in drinking water. Based on this study, applied riverbank filtration and water treatment procedures do not seem to be suitable for the total removal of estrogenic chemicals. Local contamination could play an important role in increasing the BPA content of the drinking water at the consumer endpoint.

Oxidation of Hydroxy- and Dihydroxybenzoic Acids Under the Udenfriend's Conditions. An HPLC Study.

BACKGROUND: Non-enzymatic hydroxylation of aromatic compounds to the respective phenolic derivatives is a possible metabolic pathway of xenobiotics. The formed metabolites can undergo consecutive oxidative reactions with free radicals to form potential toxic molecules. OBJECTIVE: Development of HPLC methods to separate, identify and quantitate the main products formed from salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid under in vitro hydroxylation conditions (Udenfriend's system). METHOD: An RP-HPLC-UV-Vis method was developed to separate salicylic acid and isomeric dihydroxybenzoic acids formed in the Udenfriend's system. Confirmation of structures of the oxidized products of salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was performed by HPLC-ESI-MS/MS method. RESULTS: The HPLC-UV-Vis method was evaluated for a number of validation characteristics (selectivity, repeatability and intermediate precision, LOD, LOQ and calibration range). It was found that oxidation of salicylic acid resulted in the formation of 2,3- and 2,5-dihydroxybenzoic acids. Furthermore, the hydroxylated metabolites can be further metabolized under the Udenfriend's conditions. CONCLUSION: The results give evidence for possible involvement of the oxidized metabolites of salicylic acid in the development of biological action of salicylates at the site of inflammation, where high hydroxyl radical level can be detected.

Mannich Ketones as Possible Antimycobacterial Agents.

Twenty-three known unsaturated and fused Mannich ketones and their reduced derivatives (amino alcohols) were selected for an antituberculotic study. They were screened against several mycobacterial strains including Mycobacterium tuberculosis, M. xenopi, and M. gordonae, and minimum inhibitory concentration values were also determined using the standard antituberculotic drug isoniazid (INH) as a reference. Structure-activity relationships were also studied. The mode of action of the test compounds was investigated using transmission electron microscopy, high-performance liquid chromatography, and matrix-assisted desorption/ionization mass spectrometry. Several test substances proved to be as potent as INH, but their antimycobacterial spectra were broader than that of INH. Our findings suggest that their mode of action is probably through the inhibition of mycobacterial cell wall biosynthesis.

Effect of progesterone and its synthetic analogs on reproduction and embryonic development of a freshwater invertebrate model.

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis, exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry. The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species.

Complex molecular changes induced by chronic progestogens exposure in roach, Rutilus rutilus.

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models.

Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

ß-Estradiol and ethinyl-estradiol contamination in the rivers of the Carpathian Basin.

17ß-Estradiol (E2) and 17α-ethinyl estradiol (EE2), which are environmental estrogens, have been determined with LC-MS in freshwater. Their sensitive analysis needs derivatization and therefore is very hard to achieve in multiresidue screening. We analyzed samples from all the large and some small rivers (River Danube, Drava, Mur, Sava, Tisza, and Zala) of the Carpathian Basin and from Lake Balaton. Freshwater was extracted on solid phase and derivatized using dansyl chloride. Separation was performed on a Kinetex XB-C18 column. Detection was achieved with a benchtop orbitrap mass spectrometer using targeted MS analysis for quantification. Limits of quantification were 0.05 ng/L (MS1) and 0.1 ng/L (MS/MS) for E2, and 0.001 ng/L (MS1) and 0.2 ng/L (MS/MS) for EE2. River samples contained n.d.-5.2 ng/L E2 and n.d.-0.68 ng/L EE2. Average levels of E2 and EE2 were 0.61 and 0.084 ng/L, respectively, in rivers, water courses, and Lake Balaton together, but not counting city canal water. EE2 was less abundant, but it was still present in almost all of the samples. In beach water samples from Lake Balaton, we measured 0.076-0.233 E2 and n.d.-0.133 EE2. A relative high amount of EE2 was found in river Zala (0.68 ng/L) and in Hévíz-Páhoki canal (0.52 ng/L), which are both in the catchment area of Lake Balaton (Hungary).