A synthesis on the sub-lethal toxicity of atenolol, a beta-blocker, in teleost fish.

Blood pressure medications like atenolol are detected in aquatic ecosystems. The objectives here were to (1) map the global presence of atenolol in surface water and sewage; (2) present current knowledge regarding removal efficiency and degradation of atenolol; (3) identify biological endpoints sensitive to exposure; (4) reveal molecular biomarkers that may be useful for exposure studies in fish; (5) determine whether toxicology studies are within environmental relevance. In fish, atenolol exposure affects endocrine and immune systems, metabolism, and epigenetics. Fewer than half of all studies measuring biological responses use environmentally-relevant concentrations. Heart rate appeared most sensitive to atenolol exposure relative to other endpoints. Data are lacking for behavioral responses to atenolol. Molecular biomarkers for atenolol may include those associated with acute kidney injury, cholestasis, and hypertriglyceridemia. Head kidney and liver may therefore be useful for detecting atenolol-induced effects. This review synthesizes knowledge regarding atenolol-induced toxicity in fish.

A comparative study on Fe(III)/H2O2 and Fe(III)/S2O82- systems modified by catechin for the degradation of atenolol.

The processes of Fe(III) activated persulfate (PS) and H2O2 modified by catechin (CAT) had been shown to be effective in degrading contaminants. In this study, the performance, mechanism, degradation pathways and products toxicity of PS (Fe(III)/PS/CAT) and H2O2 (Fe(III)/H2O2/CAT) systems were compared using atenolol (ATL) as a model contaminant. 91.0% of ATL degradation was reached after 60 min in H2O2 system which was much higher than that in PS system (52.4%) under the same experimental condition. CAT could react directly with H2O2 to produce small amounts of HO• and the degradation efficiency of ATL was proportional to CAT concentration in H2O2 system. However, the optimal CAT concentration was 5 µM in PS system. The performance of H2O2 system was more susceptible to pH than that of PS system. Quenching experiments were conducted indicating that SO4•- and HO• were produced in PS system while HO• and O2•- accounted for ATL degradation in H2O2 system. Seven pathways with nine byproducts and eight pathways with twelve byproducts were put forward in PS and H2O2 systems respectively. Toxicity experiments showed that the inhibition rates of luminescent bacteria were both decreased about 25% after 60 min reaction in two systems. Although the software simulation result showed few intermediate products of both systems were More toxic than ATL, but the amounts of them were 1-2 orders of magnitude lower than ATL. Moreover, the mineralization rates were 16.4% and 19.0% in PS and H2O2 systems respectively.

Lipidomic, metabolomic, and behavior responses of zebrafish (Danio rerio) exposed to environmental levels of the beta blocker atenolol.

Blood pressure medications are used to treat hypertension; however, low concentrations of beta-blockers in water systems can negatively impact aquatic wildlife. Here, we conducted a metabolic and behavioral study investigating atenolol, a beta-blocker frequently detected in global wastewater systems. The objectives were to determine the effects of low-level atenolol exposure on early stages of zebrafish. We measured survival, deformities, heartbeat, mitochondrial function, lipid and amino acid profiles, and locomotor activity to discern mechanisms of metabolic disruption. We hypothesized that atenolol disrupts lipid metabolism, which would negatively impact locomotor activity. Atenolol showed no overt toxicity to larval zebrafish up to 10 µg/L and deformities were infrequent (<5 %), and included cardiac edema and larvae with kinked tails. A hatch delay was observed at 2-day post-fertilization (dpf) for fish exposed to >5 µg/L atenolol. Heart rates were reduced in 2 and 3 dpf in fish treated with >500 ng/L atenolol. There was no change in oxygen consumption rates (basal and maximum respiration) of embryos when exposed to a range of atenolol concentrations, suggesting mitochondrial respiration was intact. Oil red staining for lipid content in larvae showed a global reduction in lipids with 10 µg/L exposure, prompting deeper investigation into the lipid profiles. Lipidomics quantified 86 lipids and revealed reduced abundance in Ceramide 18: 1 16:0 (Cer_NS d18:1_16:0), Ether linked Phosphatidylethanolamine 16:0 22:6 (EtherPE 16:0e_22:6), and Ether linked Phosphatidylcholine 16:0 22:6 (EtherPC 16:0e_22:6). We also quantified 12 amino acids and observed a subtle dose-dependent reduction in the levels of L-Histidine. Exposure to atenolol did not impact larval locomotor activity based on a Visual Motor Response test. Taken together, atenolol at environmentally relevant levels decreased heart rate of developing zebrafish and altered lipid content. As such, exposure to beta-blockers like atenolol may have negative consequences for developmental trajectories and growth of aquatic species.

In silico design and pharmacological evaluation of conjugates of atenolol with modified saccharide for cardiovascular targeting.

Amongst a wide range of biological macromolecules, saccharides exhibit the potential to be specifically recognized by cell-surface receptors and hence can be utilized as ligands in targeted drug delivery. The current study aims to use saccharides viz. Galactose, Pectin and Chitosan to improve targeting of Atenolol by oxalyl chloride mediated grafting. Conjugates were engineered by grafting Atenolol, a cardiovascular agent with the modified saccharide units. The conjugates were characterized by FTIR, DSC and 1H NMR study. Drug release analysis and cellular uptake study was carried out using H9c2 cell lines which represent that concentration of drug in cells treated with all atenolol-saccharide conjugates is enhanced by almost two-folds in comparison with cells treated with atenolol solution. Thus cell line study confers the evidence of selective cardiac delivery. No significant cytotoxicity was observed in case of all synthesized conjugates in the Brine shrimp lethality bioassay. Possible binding of the developed conjugates with the GLUT-4 receptors was assessed by in silico analysis using homology model developed by Swiss Model server. Hence it was concluded that the application of these conjugates with saccharides in selective cardiovascular drug delivery can be a promising approach to increase bioavailability, minimize drug loss by degradation and prevent harmful side effects by increasing specific cell targeting.

Toxicity of Atenolol and Propranolol on Rat Heart Mitochondria.

Propranolol and atenolol are known as ß receptor blocker drugs. These drugs are used to treat some heart diseases. There are controversies in the relationship between the use of beta-blocker drugs and the level of reactive oxygen species (ROS). Mitochondria as one of the most important sources of ROS are considered as one of the targets of drug-induced cardiotoxicity. The aim of this study was to evaluate the effects of propranolol and atenolol on mitochondria isolated from the heart. To achieve this aim, several markers of mitochondrial and cellular toxicity were evaluated. The key results of this study are the increased ROS level, collapse in mitochondrial membrane potential (MMP), mitochondrial swelling and cytochrome c release as well as disruption of respiratory chain complex II in mitochondria in isolated heart mitochondria after exposure to propranolol and atenolol. The results indicate an increase in caspase-3 activity and a decrease in the ATP level in cardiomyocytes after exposure to propranolol and atenolol. The underlying mechanisms of propranolol and atenolol induced cardiotoxicity may be associated with alterations in mitochondrial function, oxidative stress, and changes in the mitochondrial membrane.

Transcriptome analysis of the brain of the sea bream (Sparus aurata) after exposure to human pharmaceuticals at realistic environmental concentrations.

Human pharmaceuticals such as Acetaminophen, Atenolol and Carbamazepine are pseudo persistent aquatic pollutants with yet unknown sub-lethal effects at environmentally relevant concentrations. Gilthead seabream (Sparus aurata) were exposed to Acetaminophen: 31.90 ± 11.07 µg L-1; Atenolol: 0.95 ± 0.38 µg L-1 and Carbamazepine: 6.95 ± 0.13 µg L-1 in a 28 day flow through experiment to (1) determine whether exposure to low concentrations in the µg·L-1 range of the pharmaceuticals alters the brain transcriptome and, (2) identify different expression profiles and treatment specific modes of action and pathways. Despite low exposure concentrations, 411, 7 and 612 differently expressed transcripts were identified in the individual treatments with Acetaminophen, Atenolol and Carbamazepine, respectively. Functional analyses of differentially expressed genes revealed a significant over representation of several biological processes, cellular compartment features and molecular functions for both Acetaminophen and Carbamazepine treatments. Overall, the results obtained in seabream brain suggest similar physiological responses to those observed in humans also at environmental concentrations, as well as the existence of treatment specific processes that may be useful for the development of biomarkers of contamination.

Blockade of ß2-adrenoceptor, rather than ß1-adrenoceptor, deteriorates cardiac anaphylaxis in isolated blood-perfused rat hearts.

BACKGROUND: Cardiac anaphylaxis is one of the features of anaphylactic hypotension. Patients treated with propranolol, a nonselective ß-adrenoceptor (AR) antagonist, develop severe anaphylaxis, but the mechanism remains unknown. Under examination were the effects of ß1- and ß2-AR antagonist on anaphylaxis-induced coronary vasoconstriction and cardiac dysfunction in isolated blood-perfused rat hearts. METHODS: Isolated hearts from ovalbumin-sensitized Wistar rats were subjected to coronary perfusion with blood at a constant pressure and measurements were made of coronary blood flow and left ventricu-lar (LV) pressure. Following pretreatment with selective ß2-AR antagonist ICI118,551 or selective ß1-AR antagonist atenolol, cardiac anaphylaxis was induced by intracoronary injections of ovalbumin antigen. LV contractility was evaluated by the maximum increasing rate of systolic LV pressure (dP/dtmax). RESULTS: In response to antigen administrations, ICI118,551 pretreated hearts showed a greater de-crease in coronary blood flow and consequently a greater increase in coronary vascular resistance than the atenolol pretreated hearts. Pretreatment with ICI118,551 caused a greater decrease in dP/dtmax than those with atenolol. CONCLUSIONS: Cardiac anaphylaxis-induced contractile dysfunction and coronary spasm are severe in b2-, rather than ß1-AR antagonist, pretreated isolated blood-perfused rat hearts.

Hazard screening of photo-transformation products from pharmaceuticals: Application to selective ß1-blockers atenolol and metoprolol.

The identification of toxic components in cocktail mixtures of pollutants, their metabolites and transformation products (TPs) generated from environmental and treatment processes remains an arduous task. This study expanded in this area by applying a combination of chemical analytics, a battery of in vitro bioassays and an in silico "testing battery" to UV photolysis mixtures of active pharmaceutical ingredients. The objectives were to understand the toxic nature of the mixtures and to prioritize photo-TPs for risk analysis. The selective ß1-blockers Atenolol (ATL) and Metoprolol (MTL) that are ubiquitous in the aquatic environment were used as an example. The photolysis mixtures were cytotoxic to Vibrio fischeri and mammalian cells but not mutagenic in the Ames test or genotoxic in the in vitro micronucleus and umu tests. Potentially hazardous TPs were proposed by relating the observed effects to the kinetics of TP occurrence and applying in silico toxicity predictions for individual photo-TPs. This model study was done to identify principal mechanisms rather than accurately simulating environmental transformation processes. Several photo-TPs were proposed to present a greater hazard than the selected ß-blockers and therefore fate and toxicity assessments may be required to determine their environmental relevance.

Oxidative stress, energy storage, and swimming performance of Limnodynastes peronii tadpoles exposed to a sub-lethal pharmaceutical mixture throughout development.

Pharmaceutical contaminants represent emerging threats to aquatic animals and ecosystem health, and research exploring toxicological outcomes associated with these compounds in non-target wildlife has been flagged for prioritization. Amphibians represent particularly vulnerable organisms and many populations around the world are currently at risk of extinction. However, to date, relatively few studies have explored the consequences of exposures to common non-steroidal pharmaceuticals during sensitive amphibian life-stages. To address existing knowledge gaps, tadpoles of the Australian striped-marsh frog (Limnodynastes peronii) were exposed to control water and a mixture of the common pharmaceutical contaminants diclofenac, naproxen, atenolol and gemfibrozil at 0.1, 1, 10, 100 and 1000 µg/L throughout the developmental period. Effects on detoxification pathways, energy storage, growth and development, and swimming performance were assessed following exposure. Developmental rates and liver-somatic index (LSI) were significantly reduced in the highest exposure concentration, and condition factor (K) was increased at concentrations as low as 10 µg/L. Morphological endpoints were associated with significantly altered levels of hepatic triglycerides, which in turn were correlated with increased peroxidase activity in animals exposed to the highest concentration (1000 µg/L). The mixture had no significant effect on swimming performance, but a trend of decreased swimming velocity (average and maximum) was observed with increasing concentration, and this was correlated with effects on LSI. Results demonstrate that mixtures of common non-steroidal pharmaceuticals can elicit a range of physiological, metabolic and morphological responses in larval amphibians, and more research is therefore warranted to explore possible relationships between endpoints at different levels of organization.

Reactivity of ß-blockers/agonists with aqueous permanganate. Kinetics and transformation products of salbutamol.

The possible oxidation of two ß-blockers, atenolol and propranolol, and one ß-agonist, salbutamol, with aqueous potassium permanganate (KMnO4) was investigated by liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Under strong oxidation conditions (2 mg L(-1) KMnO4, 24 h), only salbutamol did significantly react. In this way, the oxidation kinetics of salbutamol was further investigated at different concentrations of KMnO4, chloride, phosphate and sample pH by means of a full factorial experimental design. Depending on these factors, half-lives were in the range 1-144 min for drug and it was observed that KMnO4 concentration was the most significant factor, resulting in increased reaction rate as it is increased. Moreover, the reaction of salbutamol is also enhanced at basic pH and to a minor extent by the presence of phosphates, being both factors more relevant at low KMnO4 concentrations. The use of an accurate-mass LC-QTOF-MS system permitted the identification of a total of seven transformation products (TPs). The transformation path of the drug begins by the attack of KMnO4 on two double bonds of the aromatic ring of salbutamol via 3 + 2 and 2 + 2 addition reactions, which resulted in the ring opening and that continues with oxidative reactions to finally produce smaller size TPs, ending with tert-butyl-formamide, as the smallest TP identified. Reaction in real samples showed a slower and partial oxidation of the pharmaceutical, due to other competing water organic constituents, but still exceeding 60%. Moreover, the software predicted toxicity of TPs indicates that they are expected not to be more toxic than salbutamol, in contrast to the results obtained for the predicted toxicity of chlorination TPs, excepting predicted developmental toxicity.

Ecotoxicity of ketoprofen, diclofenac, atenolol and their photolysis byproducts in zebrafish (Danio rerio).

The occurrence of pharmaceutical compounds in wastewater treatment plants and surface waters has been detected worldwide, constituting a potential risk for aquatic ecosystems. Adult zebrafish, of both sexes, were exposed to three common pharmaceutical compounds (atenolol, ketoprofen and diclofenac) and their UV photolysis by-products over seven days. The results show that diclofenac was removed to concentrations

The sub-lethal effects and tissue concentration of the human pharmaceutical atenolol in rainbow trout (Oncorhynchus mykiss).

Atenolol is a highly prescribed anti-hypertensive pharmaceutical and a member of the group of ß-blockers. It has been detected at concentrations ranging from ng L(-1) to low µg L(-1) in waste and surface waters. The present study aimed to assess the sub-lethal effects of atenolol on rainbow trout (Oncorhynchus mykiss) and to determine its tissue-specific bioconcentration. Juvenile rainbow trout were exposed for 21 and 42 days to three concentration levels of atenolol (1 µg L(-1) - environmentally relevant concentration, 10 µg L(-1), and 1000 µg L(-1)). The fish exposed to 1 µg L(-1) atenolol exhibited a higher lactate content in the blood plasma and a reduced haemoglobin content compared with the control. The results show that exposure to atenolol at concentrations greater than or equal to 10 µg L(-1) significantly reduces both the haematocrit value and the glucose concentration in the blood plasma. The activities of the studied antioxidant enzymes (catalase and superoxide dismutase) were not significantly affected by atenolol exposure, and only the highest tested concentration of atenolol significantly reduced the activity of glutathione reductase. The activities of selected CYP450 enzymes were not affected by atenolol exposure. The histological changes indicate that atenolol has an effect on the vascular system, as evidenced by the observed liver congestion and changes in the pericardium and myocardium. Atenolol was found to have a very low bioconcentration factor (the highest value found was 0.27). The bioconcentration levels followed the order liver>kidney>muscle. The concentration of atenolol in the blood plasma was below the limit of quantification (2.0 ng g(-1)). The bioconcentration factors and the activities of selected CYP450 enzymes suggest that atenolol is not metabolised in the liver and may be excreted unchanged.

Uptake and effects of a mixture of widely used therapeutic drugs in Eruca sativa L. and Zea mays L. plants.

Pharmaceutically active compounds (PACs) are continuously dispersed into the environment due to human and veterinary use, giving rise to their potential accumulation in edible plants. In this study, Eruca sativa L. and Zea mays L. were selected to determine the potential uptake and accumulation of eight different PACs (Salbutamol, Atenolol, Lincomycin, Cyclophosphamide, Carbamazepine, Bezafibrate, Ofloxacin and Ranitidine) designed for human use. To mimic environmental conditions, the plants were grown in pots and irrigated with water spiked with a mixture of PACs at concentrations found in Italian wastewaters and rivers. Moreover, 10× and 100× concentrations of these pharmaceuticals were also tested. The presence of the pharmaceuticals was tested in the edible parts of the plants, namely leaves for E. sativa and grains for Z. mays. Quantification was performed by liquid chromatography mass spectroscopy (LC/MS/MS). In the grains of 100× treated Z. mays, only atenolol, lincomycin and carbamazepine were above the limit of detection (LOD). At the same concentration in E. sativa plants the uptake of all PACs was >LOD. Lincomycin and oflaxacin were above the limit of quantitation in all conditions tested in E. sativa. The results suggest that uptake of some pharmaceuticals from the soil may indeed be a potential transport route to plants and that these environmental pollutants can reach different edible parts of the selected crops. Measurements of the concentrations of these pharmaceuticals in plant materials were used to model potential adult human exposure to these compounds. The results indicate that under the current experimental conditions, crops exposed to the selected pharmaceutical mixture would not have any negative effects on human health. Moreover, no significant differences in the growth of E. sativa or Z. mays plants irrigated with PAC-spiked vs. non-spiked water were observed.

Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus.

Pharmaceuticals are widely used in human and veterinary medicine. However, they are emerging as a significant contaminant in aquatic environments through wastewater. Due to the persistent and accumulated properties of pharmaceuticals via the food web, their potential harmful effects on aquatic animals are a great concern. In this study, we investigated the effects of six pharmaceuticals: acetaminophen, ATP; atenolol, ATN; carbamazepine, CBZ; oxytetracycline, OTC; sulfamethoxazole, SMX; and trimethoprim, TMP on acetylcholinesterase (AChE; EC 3.1.1.7) activity and its transcript expression with chlorpyrifos (as a positive control) in the monogonont rotifer, Brachionus koreanus. ATP, CBZ, and TMP exposure also remarkably inhibited Bk-AChE activity at 100 µg/L (24 h) and 1000 µg/L (12 h and 24 h). ATP, CBZ, and TMP exposure showed a significant decrease in the Bk-AChE mRNA level in a concentration-dependent manner. However, in the case of OTC and SMX, a slight decrease in Bk-AChE mRNA expression was found but only at the highest concentration. The time-course experiments showed that ATP positively induced Bk-AChE mRNA 12 h after exposure at both 100 and 1000 µg/L, while the Bk-AChE mRNA expression was significantly downregulated over 6 to 24 h after exposure to 1000 µg/L of CBZ, OTC, SMX, and TMP. Our findings suggest that Bk-AChE would be a useful biomarker for risk assessment of pharmaceutical compounds as an early signal of their toxicity in aquatic environments. Particularly, ATP, CBZ, and TMP may have a toxic cholinergic effect on rotifer B. koreanus by inhibiting AChE activity.

Simultaneous monitoring of photocatalysis of three pharmaceuticals by immobilized TiO2 nanoparticles: chemometric assessment, intermediates identification and ecotoxicological evaluation.

In this study, the photocatalytic degradation of a mixture of three pharmaceuticals, Metronidazole (MET), Atenolol (ATL) and Chlorpromazine (CPR), was quantified simultaneously during the UV/TiO2 process. The investigated TiO2 was Millennium PC-500 immobilized on ceramic plates by sol-gel based method. The partial least squares modeling was successfully applied for the multivariate calibration of the spectrophotometric data. The central composite design was applied to model and optimize the UV/TiO2 process. Predicted values of removal efficiency were found to be in good agreement with experimental values for MET, ATL and CPR (R(2)=0.947 and Adj-R(2)=0.906, R(2)=0.977 and Adj-R(2)=0.960 and R(2)=0.982 and Adj-R(2)=0.969, respectively). The optimum initial concentration of pharmaceuticals, reaction time and UV light intensity was found to be 10 mg L(-1), 150 min and 38.45 W m(-2), respectively. The main degradation intermediates of pharmaceuticals produced in this process were identified by GC-MS technique. The chronic ecotoxicity of pharmaceuticals was evaluated using aquatic species Spirodela polyrrhiza prior to and after photocatalysis. The TOC results (90% removal after 16 h) and ecotoxicological experiments revealed that the photocatalysis process could effectively mineralize and reduce the ecotoxicity of the pharmaceuticals from their aqueous solutions.

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor-based assay for monitoring the drug atenolol.

Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive-binding assay, based on the interaction between ATL and its target receptor, ß1 adrenergic receptor (ß1AR). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme-linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 ± 0.048 and 0.032 ± 0.016 ng/ml, respectively, and the Abs did not cross-react with any of the tested beta-blocker drugs. Furthermore, a human ß1AR (h-ß1AR) was stably expressed in Spodoptera frugiperda cells (Sf9). The receptor was employed to develop a competitive-binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h-ß1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose-dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost-effective large-scale, high-throughput monitoring of ATL in environmental, agricultural, and biological samples.

Nitrate-induced photodegradation of atenolol in aqueous solution: kinetics, toxicity and degradation pathways.

The extensive utilization of ß-blockers worldwide led to frequent detection in natural water. In this study the photolysis behavior of atenolol (ATL) and toxicity of its photodegradation products were investigated in the presence of nitrate ions. The results showed that ATL photodegradation followed pseudo-first-order kinetics upon simulated solar irradiation. The photodegradation was found to be dependent on nitrate concentration and increasing the nitrate from 0.5 mML(-1) to 10 mML(-1) led to the enhancement of rate constant from 0.00101 min(-1) to 0.00716 min(-1). Hydroxyl radical was determined to play a key role in the photolysis process by using isopropanol as molecular probe. Increasing the solution pH from 4.8 to 10.4, the photodegradation rate slightly decreased from 0.00246 min(-1) to 0.00195 min(-1), probably due to pH-dependent effect of nitrate-induced .OH formation. Bicarbonate decreased the photodegradation of ATL in the presence of nitrate ions mainly through pH effect, while humic substance inhibited the photodegradation via both attenuating light and competing radicals. Upon irradiation for 240 min, only 10% reduction of total organic carbon (TOC) can be achieved in spite of 72% transformation rate of ATL, implying a majority of ATL transformed into intermediate products rather than complete mineralization. The main photoproducts of ATL were identified by using solid phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) techniques and possible nitrate-induced photodegradation pathways were proposed. The toxicity of the phototransformation products was evaluated using aquatic species Daphnia magna, and the results revealed that photodegradation was an effective mechanism for ATL toxicity reduction in natural waters.

Venlafaxine and atenolol disrupt epinephrine-stimulated glucose production in rainbow trout hepatocytes.

The beta-blocker atenolol (ATEN), and the selective serotonin and norepinephrine reuptake inhibitor, venlafaxine (VEN) are found in municipal wastewater effluents, but little is known about the effect of these pharmaceuticals on aquatic animals. We tested the hypothesis that VEN and ATEN disrupt acute stress mediated glucose production in fish liver. To this end, rainbow trout (Oncorhynchus mykiss) hepatocytes were exposed in vitro to different concentrations (0, 0.1, 10, 1000 nM) of VEN or ATEN and glucose production in response to either cortisol or epinephrine (two key stress hormones) was ascertained. Both VEN and ATEN did not affect either the unstimulated or cortisol (100 ng/mL)-stimulated glucose release over a 24 h period. The acute (3 h) unstimulated glucose production by isolated hepatocytes in suspension was also not modified by ATEN, while VEN (100 and 1000 nM) reduced basal glucose release. However, ATEN, even at concentration as low as 0.01 nM completely abolished epinephrine (1 µM)-induced glucose production in trout hepatocytes. Interestingly, VEN also suppressed epinephrine-induced glucose production but only at higher concentrations (100 and 1000 nM). Neither VEN nor ATEN significantly impacted the glucose production in response to either 8-bromo-cAMP (cAMP analogue) or glucagon (a metabolic hormone that increases glucose production) stimulation. ATEN but not VEN attenuated the epinephrine-induced increase in glucose transporter 2 (GLUT2) mRNA abundance in trout hepatocytes. Taken together, our results suggest that the impact of ATEN and VEN on glucose production involves inhibition of ß-adrenoceptor signaling in trout hepatocytes. Overall, VEN and ATEN are beta-blockers and may disrupt the adaptive acute glucose response to a secondary stressor in rainbow trout.

Cytotoxicity assessment of four pharmaceutical compounds on the zebra mussel (Dreissena polymorpha) haemocytes, gill and digestive gland primary cell cultures.

Pharmaceutical compounds are considered the new environmental pollutants but at present few studies have evaluated their ecotoxicity on aquatic invertebrates. This study was aimed to investigate the in vitro cytotoxicity of four common drugs, namely atenolol (ATL), carbamazepine (CBZ), diclofenac (DCF) and gemfibrozil (GEM), on three different cell typologies from the zebra mussel (Dreissena polymorpha): haemocytes, gill and digestive gland cells. Results obtained by the Trypan blue exclusion test revealed that exposure to increasing concentrations (0.001; 0.01; 0.1; 1 and 10 mg L(-1)) of CBZ, DCF and GEM were able to significantly decrease the viability of each cell type, while the MTT (3(4,5-dimethyl-2thiazholyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay highlighted only a slight reduction of mitochondrial activity of gill and digestive gland cells. Overall, DCF was the most cytotoxic drug for zebra mussel cells, followed by GEM, CBZ, while ATL has not a noteworthy toxic potential. Our preliminary results lay the groundwork for further in vitro evaluations, which will allow a better definition of the potential toxicity of these drugs.

Potential physiological effects of pharmaceutical compounds in Atlantic salmon (Salmo salar) implied by transcriptomic analysis.

BACKGROUND, AIM, AND SCOPE: Pharmaceuticals are emerging pollutants widely used in everyday urban activities which can be detected in surface, ground, and drinking waters. Their presence is derived from consumption of medicines, disposal of expired medications, release of treated and untreated urban effluents, and from the pharmaceutical industry. Their growing use has become an alarming environmental problem which potentially will become dangerous in the future. However, there is still a lack of knowledge about long-term effects in non-target organisms as well as for human health. Toxicity testing has indicated a relatively low acute toxicity to fish species, but no information is available on possible sublethal effects. This study provides data on the physiological pathways involved in the exposure of Atlantic salmon as representative test species to three pharmaceutical compounds found in ground, surface, and drinking waters based on the evaluation of the xenobiotic-induced impairment resulting in the activation and silencing of specific genes. MATERIALS AND METHODS: Individuals of Atlantic salmon (Salmo salar) parr were exposed during 5 days to environmentally relevant concentrations of three representative pharmaceutical compounds with high consumption rates: the analgesic acetaminophen (54.77+/-34.67 microg L(-1)), the anticonvulsant carbamazepine (7.85+/-0.13 microg L(-1)), and the beta-blocker atenolol (11.08+/-7.98 microg L(-1)). Five immature males were selected for transcriptome analysis in brain tissues by means of a 17k salmon cDNA microarray. For this purpose, mRNA was isolated and reverse-transcribed into cDNA which was labeled with fluorescent dyes and hybridized against a common pool to the arrays. Lists of significantly up- and down-regulated candidate genes were submitted to KEGG (Kyoto Encyclopedia of Genes and Genomes) in order to analyze for induced pathways and to evaluate the usefulness of this method in cases of not completely annotated test organisms. RESULTS: Exposure during 5 days to environmentally relevant concentrations of the selected pharmaceutical compounds acetaminophen, carbamazepine, and atenolol produced differences in the expression of 659, 700, and 480 candidate genes, respectively. KEGG annotation numbers (KO annotations) were obtained for between 26.57% and 33.33% of these differently expressed genes per treatment in comparison to non-exposure conditions. Pathways that showed to be induced did not always follow previously reported targets or metabolic routes for the employed treatments; however, several other pathways have been found (four or more features) to be significantly induced. DISCUSSION: Energy-related pathways have been altered under exposure in all the selected treatments, indicating a possible energy budget leakage due to additional processes resulting from the exposure to environmental contaminants. Observed induction of pathways may indicate additional processes involved in the mode of action of the selected pharmaceuticals which may not have been detected with conventional methods like quantitative PCR in which only suspected features are analyzed punctually for effects. The employment of novel high-throughput screening techniques in combination with global pathway analysis methods, even if the organism is not completely annotated, allows the examination of a much broader range of candidates for potential effects of exposure at the gene level. CONCLUSIONS: The continuously growing number of annotations of representative species relevant for environmental quality testing is facilitating pathway analysis processes for not completely annotated organisms. KEGG has shown to be a useful tool for the analysis of induced pathways from data generated by microarray techniques with the selected pharmaceutical contaminants acetaminophen, carbamazepine, and atenolol, but further studies have to be carried out in order to determine if a similar expression pattern in terms of fold change quantity and pathways is observed after long-term exposure. Together with the information obtained in this study, it will then be possible to evaluate the potential risk that the continuous release of these compounds may have on the environment and ecosystem functioning.