Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors.

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to ß-naphthoflavone (ß-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to ß-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Physiological and metabolic alterations induced by commercial neonicotinoid formulations in Daphnia magna.

Neonicotinoid insecticides are widely used agents in agriculture to control a broad range of insect pests. Although use of neonicotinoid pesticides has resulted in the widespread contamination of surface waters, sublethal toxicity data of these products in relation to non-target aquatic biota are still poor. Therefore, the objective of this study was to assess the effects of two neonicotinoid pesticides with widespread use on the basic physiological functions: the thoracic limb activity and heart rate of Daphnia magna, and to screen for their potential to affect the cytochrome P450 monooxygenase system (ECOD activity) of daphnids. The considered pesticides were the acetamiprid- and thiacloprid based products Mospilan 20 SG and Calypso 480 SC, respectively. The dose-dependent variation in the three biological endpoints considered were assessed following 24 h exposures. The two neonicotinoid formulations elicited significant depression on the thoracic limb activity and heart rate of daphnids at doses close to the immobility thresholds of formulations (48h-EC50: Mospilan 20 SG = 190 mg L-1; Calypso 480 SC = 120 mg L-1), an effect mainly attributable to the overall drop in the general health status of the organisms. The alterations in the physiological traits were significant at exposures to 190 mg L-1 for Mospilan 20 SG and 48 mg L-1 for Calypso 480 SC. The dose related variation in the ECOD activity of daphnids exposed to the selected neonicotinoid formulations followed a biphasic pattern, with starting effective doses for Mospilan 20 SG of 6.3 mg L-1 (=1/20 of 48h-EC50 for Daphnia neonates), and for Calypso 480 SC of 0.034 mg L-1 (=1/4000 of 48h-EC50). Maximal ECOD activity (2.2 fold increase vs. controls) was induced by Mospilan 20 SG in daphnids exposed to 114 mg L-1 product (=48 h-EC20), and by Calypso 480 SC (1.8 fold increase) at 5.2 mg L-1 dose (=1/20 of 48 h-EC50). Our results outlined significant alterations in the physiological traits and ECOD activity in exposed daphnids at concentrations below the immobility thresholds (48 h-EC50) of the products used as benchmarks to rate their toxicity risks to aquatic biota. Therefore, we think our findings might deserve consideration in the environmental risk evaluation of these products.

Modeling and mitigating supply chain disruptions as a bilevel network flow problem.

Years of globalization, outsourcing and cost cutting have increased supply chain vulnerability calling for more effective risk mitigation strategies. In our research, we analyze supply chain disruptions in a production setting. Using a bilevel optimization framework, we minimize the total production cost for a manufacturer interested in finding optimal disruption mitigation strategies. The problem constitutes a convex network flow program under a chance constraint bounding the manufacturer's regrets in disrupted scenarios. Thus, in contrast to standard bilevel optimization schemes with two decision-makers, a leader and a follower, our model searches for the optimal production plan of a manufacturer in view of a reduction in the sequence of his own scenario-specific regrets. Defined as the difference in costs of a reactive plan, which considers the disruption as unknown until it occurs, and a benchmark anticipative plan, which predicts the disruption in the beginning of the planning horizon, the regrets allow measurement of the impact of scenario-specific production strategies on the manufacturer's total cost. For an efficient solution of the problem, we employ generalized Benders decomposition and develop customized feasibility cuts. In the managerial section, we discuss the implications for the risk-adjusted production and observe that the regrets of long disruptions are reduced in our mitigation strategy at the cost of shorter disruptions, whose regrets typically stay far below the risk threshold. This allows a decrease of the production cost under rare but high-impact disruption scenarios.

Reference data for ultrasound bone characteristics in Hungarian children aged 7-19 years.

Backround: Osteoporosis is a common disease and physical activity (PA) has a favourable influence on bone status. AIM: To establish normative data for calcaneal quantitative ultrasound (QUS) bone characteristics in children and to analyse the relationships between PA, anthropometric and bone parameters. SUBJECTS AND METHODS: Hungarian children aged 7-19 (n = 2674; 1325 girls, 1349 boys) provided PA, anthropometric and bone data. QUS parameters were registered with Sonost3000 densitometer (speed of sound: SOS, m/s; broadband ultrasound attenuation: BUA, dB/MHz; bone quantity index (BQI = αSOS + ßBUA). Reference centiles of QUS parameters were constructed by LMS method. Multivariate linear regression models were used to analyse the relationships. RESULTS: QUS bone parameters increased with age. There were no gender differences, except in 11- and 19-year-old children, where boys showed higher values. SOS (1497.15 ± 15.72 vs 1494.05 ± 14.81 m/s) and BQI (65.31 ± 16.71 vs 62.26 ± 15.78) were higher in athletic children. The regression model revealed significant relations between SOS and age, relative muscle mass and PA; BUA correlated with gender, morphological age, BMI, relative muscle mass and PA. CONCLUSIONS: Changes in the bone parameters among 7-19-year-old Hungarian children depended on age, anthropometric dimensions and the level of PA. The normative data could be used for monitoring QUS bone parameters in children, leading to more effective interventions for healthy bones.

Inhibitory effects of four neonicotinoid active ingredients on acetylcholine esterase activity.

There is a great concern about the decline of pollinators, and neonicotinoids emerging bee disorders are assumed to play a significant role. Since changes in learning ability has been observed in honey bees exposed to some acetylcholine esterase (AChE) inhibitors, we therefore, tested in vitro the effect of four neonicotinoids on purified eel AChE. AChE activity was inhibited in a concentration-dependent manner, and calculated IC50 values for thiamethoxam (IC50 = 414 µM) and clothianidin (IC50 = 160 µM) were found to be much higher compared to acetamiprid (IC50 = 75.2 µM) and thiacloprid (IC50 = 87.8 µM). The Lineweaver-Burk reciprocal plots for acetamiprid shows unchanged Vmax and increased Km values with inhibitor concentrations, while analysis of Michaelis-Menten plots shows predominantly competitive mechanism. The inhibition constant value (Ki = 24.3 µM) indicates strong binding of the acetamiprid complex to AChE. Finally, the four tested neonicotinoids are not a uniform group regarding their blocking ability. Our results suggest a previously not established, direct AChE blocking mechanism of neonicotinoids tested, thus the neuronal AChE enzyme is likely among the direct targets of the neonicotinoid insecticides. We conclude, that these AChE inhibitory effects may also contribute to toxic effects on the whole exposed animal.

Temperature related toxicity features of acute acetamiprid and thiacloprid exposure in Daphnia magna and implications on reproductive performance.

This study investigated the potential for elevated temperature to alter the toxicity of acetamiprid (ACE) and thiacloprid (Thia) in the ecotoxicity model Daphnia magna. The modulation of CYP450 monooxygenases (ECOD), ABC transporter activity (MXR) and incident cellular reactive oxygen species (ROS) overproduction was screened in premature daphnids following acute (48 h) exposure to sublethal concentrations of ACE and Thia (0.1-, 1.0 µM) at standard 21 °C and elevated 26 °C temperatures. Delayed outcomes of acute exposures were further evaluated based on the reproduction performance of daphnids monitored over 14 days of recovery. Exposures to ACE and Thia at 21o C elicited moderate induction of ECOD activity, pronounced inhibition of MXR activity and severe ROS overproduction in daphnids. In the high thermal regime, treatments resulted in significantly lower induction of ECOD activity and inhibition of MXR activity, suggesting a suppressed metabolism of neonicotinoids and less impaired membrane transport activity in daphnids. Elevated temperature on its own, caused a three-fold rise in ROS levels in control daphnids, while ROS overproduction upon neonicotinoid exposure was less accentuated. Acute exposures to ACE and Thia caused significant decreases also in the reproduction of daphnids, indicating delayed outcomes even at environmentally relevant concentrations. Both the cellular alterations in exposed daphnids and decreases in their reproductive output post exposures evidenced closely similar toxicity patterns and potentials for the two neonicotinoids. While elevated temperature elicited only a shift in baseline cellular alterations evoked by neonicotinoids, it significantly worsened the reproductive performance of daphnids following neonicotinoid exposures.

Utilizing additive manufacturing and mass customization under capacity constraints.

Additive manufacturing (AM), originally used for prototyping, is increasingly adopted for custom final part production across different industries. However, printing speed and production volume are two barriers for the adoption of AM for product customization at large scale. Nevertheless, manufacturers could aim to combine the benefits of AM for product customization with traditional mass customization (MC) technologies over the product life cycle (PLC). This approach is showcased in our paper as a manufacturing opportunity and is addressed via a non convex-concave optimization model that considers a monopolist manufacturer producing horizontally differentiated products at scale. To satisfy individual customer preferences under capacity considerations, the firm jointly decides on the inventory, production quantity, product variety, optimal technology-switching times (between AM and MC) and pricing strategy. Our approach can be implemented by decision-makers to leverage customer-centricity and benefit from this novel hybrid manufacturing practice. By deriving a closed-form solution for the production quantity based on an adaptive inventory policy, the resulting optimization problem is solved using the Sample Average Approximation framework grounded by analytical results. Our results demonstrate that the new usage of AM with MC can benefit a manufacturer for customer-centric driven strategies. Significant profit improvements can be achieved with an AM-MC-AM technology-switching scenario under certain capacity conditions and with an increasing-decreasing pricing strategy. Our results also indicate that the benefits of pricing flexibility are highest when capacity is unlimited or when the firm does not hold inventory. Under capacity constraints, a simple decreasing pricing policy combined with inventory performs very well.

Presence, variation, and potential ecological impact of microplastics in the largest shallow lake of Central Europe.

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.

Neurotoxic effects evoked by cyanobacterial extracts suggest multiple receptors involved in electrophysiological responses of molluscan (CNS, heart) models.

The responses of the snail central neurons (Helix pomatia, Lymnaea stagnalis) and the isolated Helix heart were characterized evoked by cyanobacterial extracts (Cylindrospermopsis raciborskii ACT strains) isolated from Lake Balaton (Hungary). The nicotinergic acetylcholine (ACh) receptors in the CNS (both excitatory and inhibitory) were blocked by the extracts of ACT 9502 and ACT 9505 strains and the anatoxin- a (homoanatoxin-a) producing reference strain of Oscillatoria sp. (PCC 6506), similar to the inhibitory effects of the pure anatoxin-a. The enhancement of the ACh responses by the ACT 9504 extract suggests additional, probably acetylcholine esterase inhibitory mechanisms. On the isolated Helix heart the crude ACT 9505 and PCC 6506 extracts evoked frequency increase and transient twitch contraction, opposite to the ACh evoked heart relaxation. Anatoxin-a similarly contracted the heart but did not increase its contration frequency. These data suggest the involvement of some non-cholinergic mechanisms, acting very likely by direct modulation of the electrical or contractile system of the isolated heart. Diversity of the effects evoked by the cyanobacterial extracts in the CNS and heart suggest pharmacologically different neuroactive components among the secondary metabolites of the cyanobacteria acting on both (anatoxin-a like) cholinergic and (unidentified) non-cholinergic receptors.

Secular growth trend characteristics of the body structure in Hungarian athlete boys.

The aim of the present study was to get more knowledge of the nature of athlete boy's secular body changes and to differentiate it from the individual acceleration. A positive secular trend was supposed in athlete youth similar to normal population, but with different extent because of the sport selection. In this study some specific trend characteristics of athlete (basketball player) boys between the 1990th and 2010th are analyzed and compared to that of the reference values of the Hungarian National Growth Study I and II (HNGS I - 1980's, Eiben et al. 1991; HNGS II - 2003-2006, Bodzsár & Zsákai 2007). The individual data were collected in Budapest (capital) basketball club boys aged 10-15 years (N = 1376). Body measurements were taken by the first author by the request of coaches and parents. Secular trend was characterized by the changes in some of the measured body parameters by three birth cohorts. The secular changes followed a positive trend, though the traditional decade analyses the variables did not follow a balanced, proportional manner of change. The largest difference in body height occurred in 12-years of age, with a total of +8.27 cm height difference between Cohort III and I and +9.31 kg total difference in body mass at the same age, respectively. Some differences in the tendentious changes occurred between the age groups, characteristic shift of the fat content and the trunk/extremity fat ratio increase toward the younger age, and more excessive increase in BMI were found. Though it could be expected to have favourable body composition in young athletes, nowadays some unfavourable changes in the secular growth trend of body structure have been occurred. One explanation could be the less active leisure time spent. Our results can provide more practical information for coaches to enrich their professional attitude towards a better understanding how to choose and modify training methods during the varied growth and developmental process of young athletes.

Behavioral and biochemical alterations induced by acute clothianidin and imidacloprid exposure in the killer shrimp, Dikerogammarus villosus.

Neonicotinoids are widely used insecticides around the world and are preserved permanently in soils and appear in surface waters posing an increased threat to ecosystems. In the present study, we exposed adult specimens of amphipod Dikerogammarus villosus to environmentally relevant and higher concentrations of two widely used agricultural neonicotinoids, clothianidin (CLO) and imidacloprid (IMI), for 2 days. The acute effects were investigated at the behavioral (immobility time and swimming activity) and biochemical (glutathione S-transferase [GST] and acetylcholine esterase [AchE] activity) levels. All CLO concentrations used (64 nM, 128 nM, 192 nM) significantly decreased the immobility time and swimming activity. In the case of IMI, the immobility time decreased significantly only at the highest concentration applied (977 nM), but the distance travelled by the animals significantly decreased even at lower concentrations (78 nM and 313 nM). The GST enzyme activity did not change in the CLO-treated groups, however, the 626 nM and 977 nM IMI concentrations significantly increased the GST activity. Similarly, to the behavioral level, all CLO concentrations significantly decreased the AchE activity. In contrast, IMI has a significant stimulating effect on the AchE activity at the 313 nM, 626 nM, and 977 nM concentrations. Based on the authors' best knowledge, this is the first study to investigate the effects of CLO and IMI at environmentally-relevant concentrations on D. villosus. Our findings contribute to the understanding of the physiological effects of neonicotinoids.

Effects of rotenone and other mitochondrial complex I inhibitors on the brine shrimp Artemia.

(Artemia) nauplii was used to asses the toxicity of rotenone, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), MP+ (1-methyl-4-phenylpyridinium) and the effect of L-DOPA co-treatment with rotenone. Rotenone had a dose dependent effect on mortality (LC50: 0.37 ± 0.04 µM mean ± S E, n = 24), while MPTP and MP+ proved to be toxic in millimolar range (LC50: 0.21 ± 0.09 mM and 0.20 ± 0.08 mM, respectively, n = 4). L-DOPA (50-200 µM) co-treatment increased the survival of the rotenone-treated animals (LC50: 0.51 ± 0.23 µM, 1.03 ± 0.66 µM, and 0.76 ± 0.52 µM, respectively). In the whole body tissue homogenates of Artemia, sublethal (up to 0.3 µM) concentrations of rotenone increased the glutathione S-transferase (GST) activity by up to 50 about percent (LC50: 53.3 ± 6.8 nM/min/mg protein, against 34.7 ± 3.6 nM/min/mg protein, n = 4). Nauplii treated in 100 mM L-DOPA and rotenone together showed further increase of GST activity all across the range of rotenone concentrations. These results on Artemia nauplii show similarities with other animal models, when complex I inhibitors were tested. Biochemical measurements suggest a protective role of L-DOPA by increasing the GST activity as part of the intracellular defences during toxin-evoked oxidative stress.

Evaluation of Possible Processing Time Effects on the Global N-Glycosylation Profile of Human Blood Samples.

The utilization of N-glycan profiling recently gained high importance in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In this paper, the effect of human blood sample handling was studied prior to N-glycosylation profiling by capillary electrophoresis, coupled with high sensitivity fluorescence detection. Special attention was paid to the preservation of sialylated structures because of their important clinical - biological relevance. Our results suggested that it is adequate to refrigerate and store the collected total blood samples prior to analysis to obtain unbiased results. Furthermore, we report on the good practice of serum sample handling in order to prevent decomposition of the sialylated structures. Our findings may promote procedure standardization and easier clinical translation of diagnostic N-glycosylation profiling in molecular medicinal applications.

Multi-marker approach for the evaluation of environmental impacts of APACS 50WG on aquatic ecosystems.

Neonicotinoids are the most widely used synthetic insecticides in the world. These insecticides are widely distributed in the ecosystem, indicating that more attention should be paid to the potential risks regarding their use in agriculture. Due their intensive use, non-target species in the environment are also exposed to their putative effects. Within acute exposure trials, the time related effect of sublethal dose of the neonicotinoid preparation APACS 50 WG was investigated on swimming behaviour and the multi-xenobiotic resistance system (MXR) activity, as a first line defence pathway of adult Dikerogammarus villosus. Results showed that treated animals manifested an increased swimming activity. Exposed animals were monitored by the rhodamine B accumulation assay, and APACS 50 WG exerted distinct changes in the MXR activity as well. Our results suggested that application of neonicotinoid at a low concentration (3.9 ng/l) contributed to the activation of locomotor activity and at the same concentration range the transmembrane transport mechanisms was altered too.

Comparative analysis of the human serum N-glycome in lung cancer, COPD and their comorbidity using capillary electrophoresis.

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to their comorbidity. In this pilot study the N-glycosylation profile of pooled human serum samples (90 patients each) from lung cancer, COPD and comorbidity (LC with COPD) patients were investigated in comparison to healthy individuals (control) by capillary gel electrophoresis with high sensitivity laser-induced fluorescence detection. Sample preparation was optimized for human serum samples introducing a new temperature adjusted denaturation protocol to prevent precipitation and increased endoglycosidase digestion time to assure complete removal of the N-linked carbohydrates. The reproducibility of the optimized method was <3.5%. Sixty-one N-glycan structures were identified in the pooled control human serum sample and the profile was compared to pooled lung cancer, COPD and comorbidity of COPD with lung cancer patient samples. One important finding was that no other sugar structures were detected in any of the patient groups, only quantitative differences were observed. Based on this comparative exercise, a panel of 13 N-glycan structures were identified as potential glycobiomarkers to reveal significant changes (>33% in relative peak areas) between the pathological and control samples. In addition to N-glycan profile changes, alterations in the individual N-glycan subclasses, such as total fucosylation, degree of sialylation and branching may also hold important glycobiomarker values.

Modeling of the Desialylated Human Serum N-glycome for Molecular Diagnostic Applications in Inflammatory and Malignant Lung Diseases.

BACKGROUND: Immunoglobulin G and A, transferrin, haptoglobin and alpha-1- antitrypsin represent approximately 85% of the human serum glycoproteome and their N-glycosylation analysis may lead to the discovery of important molecular disease markers. However, due to the labile nature of the sialic acid residues, the desialylated subset of the serum N-glycoproteome has been traditionally utilized for diagnostic applications. OBJECTIVE: Creating a five-protein model to deconstruct the overall N-glycosylation fingerprints in inflammatory and malignant lung diseases. METHODS: The N-glycan pool of human serum and the five high abundant serum glycoproteins were analyzed. Simultaneous endoglycosidase/sialidase digestion was followed by fluorophore labeling and separation by CE-LIF to establish the model. Pooled serum samples from patients with COPD, lung cancer (LC) and their comorbidity were all analyzed. RESULTS: Nine significant (>1%) asialo-N-glycan structures were identified both in human serum and the standard protein mixture. The core-fucosylated-agalacto-biantennary glycan differentiated COPD and LC and both from the control and the comorbidity groups. Decrease in the core-fucosylated-agalacto-biantennary-bisecting, monogalacto and bigalacto structures differentiated all disease groups from the control. The significant increase of the fucosylated-galactosylated-triantennary structure was highly specific for LC, to a medium extent for COPD and a lesser extent for comorbidity. Also, some increase in the afucosylated-galactosylated-biantennary structure in all three disease types and afucosylated-galactosylated-triantennary structures in COPD and LC were observed in comparison to the control group. CONCLUSION: Our results suggested that changes in the desialylated human serum Nglycome hold glycoprotein specific molecular diagnostic potential for malignant and inflammatory lung diseases, which can be modeled with the five-protein mixture.

Embryo/larval toxicity and transcriptional effects in zebrafish (Danio rerio) exposed to endocrine active riverbed sediments.

Sediment toxicity plays a fundamental role in the health of inland fish communities; however, the assessment of the hazard potential of contaminated sediments is not a common objective in environmental diagnostics or remediation. This study examined the potential of transcriptional endpoints investigated in zebrafish (Danio rerio) exposed to riverbed sediments in ecotoxicity testing. Embryo-larval 10-day tests were conducted on sediment samples collected from five sites (one upstream and four downstream of the city of Milan) along a polluted tributary of the Po River, the Lambro River. Sediment chemistry showed a progressive downstream deterioration in river quality, so that the final sampling site showed up to eight times higher concentrations of, for example, triclosan, galaxolide, PAH, PCB, BPA, Ni, and Pb, compared with the uppermost site. The embryo/larval tests showed widespread toxicity although the middle river sections evidenced worse effects, as evidenced by delayed embryo development, hatching rate, larval survival, and growth. At the mRNA transcript level, the genes encoding biotransformation enzymes (cyp1a, gst, ugt) showed increasing upregulations after exposure to sediment from further downstream sites. The genes involved in antioxidant responses (sod, gpx) suggested that more critical conditions may be present at downstream sites, but even upstream of Milan there seemed to be some level of oxidative stress. Indirect evidences of potential apoptotic activity (bcl2/bax < 1) in turn suggested the possibility of genotoxic effects. The genes encoding for estrogen receptors (erα, erß1, erß2) showed exposure to (xeno)estrogens with a progressive increase after exposure to sediments from downstream sites, paralleled by a corresponding downregulation of the ar gene, likely related to antiandrogenic compounds. Multiple levels of thyroid disruption were also evident particularly in downstream zebrafish, as for thyroid growth (nkx2.1), hormone synthesis and transport (tg, ttr, d2), and signal transduction (trα, trß). The inhibition of the igf2 gene reasonably reflected larval growth inhibitions. Although none of the sediment chemicals could singly explain fish responses, principal component analysis suggested a good correlation between gene transcripts and the overall trend of contamination. Thus, the combined impacts from known and unknown covarying chemicals were proposed as the most probable explanation of fish responses. In summary, transcriptional endpoints applied to zebrafish embryo/larval test can provide sensitive, comprehensive, and timeliness information which may greatly enable the assessment of the hazard potential of sediments to fish, complementing morphological endpoints and being potentially predictive of longer studies.

Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells.

In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

Polybrominated diphenyl ethers (PBDEs) in gammarids, caddisflies, and bed sediments of the lowland River Po.

Polybrominated diphenyl ethers (PBDEs) were investigated in sediments and invertebrates (gammarids and caddisflies) collected in the River Po, upstream and downstream from a polluted tributary. Besides a diffuse contamination by penta-BDE technical mixture, the river sediments identified the tributary as an important source to the main river of decabromodiphenyl ether (BDE-209), which peaked to 64 microg/g (OC) in the downstream stretch. At 10 km downstream from the tributary, a higher bioavailability was evident than at 22 km, and small gammarids accumulated at two/three times the levels of PBDEs found in large gammarids. The congener profiles of sediments and invertebrates were dominated by BDE-47, BDE-99 and BDE-209.

The allelochemical tannic acid affects the locomotion and feeding behaviour of the pond snail, Lymnaea stagnalis, by inhibiting peripheral pathways.

(1) The effect of tannic acid (TA), a dominant component of plant allelochemicals, was investigated on the locomotion and feeding of the pond snail, Lymnaea stagnalis. The effect of TA on the neuronal background underlying feeding activity was also analysed. (2) TA affected the spontaneous locomotion and of juvenile snails in a concentration-dependent way. Low (10 µM) TA concentration resulted in an increased (sliding or swimming) activity compared to the control; meanwhile, high (100 µM) TA concentration inhibited the locomotion of the animals. (3) Low (10 µM) TA concentration increased the frequency of sucrose-evoked feeding of intact animals, whereas high (100 µM) TA concentration resulted in significantly longer feeding latency and decreased feeding rate. The feeding changes proved to be partially irreversible, since after 48 h maintained in clear water, the animals tested in 100 µM TA previously still showed lower feeding rate in sucrose. (4) Electrophysiological experiments on semi-intact preparations showed that application of 100 µM TA to the lip area inhibited the fictive feeding pattern of central neurons, the cellular response to sucrose. (5) On isolated CNS preparation, 100 µM TA applied in the bathing solution, however, failed to inhibit the activation of the central feeding (CPG) interneurons following application of extracellular dopamine. Our results suggest that TA affects both afferent and efferent peripheral functions in Lymnaea. TA reduces feeding activity by primarily blocking feeding sensory pathways, and its negative effect on locomotion may imply sensory pathways and/or ciliary activity.