Inflammatory properties of municipal effluents to Elliptio complanata mussels--lack of effects from anti-inflammatory drugs

The purpose of this study was to identify the pharmacological effects of anti-inflammatory drugs in freshwater mussels (Elliptio complanata) exposed to a primary-treated municipal effluent. Mussel specimens were injected with either increasing concentrations of ibuprofen or with a municipal effluent extract, and then left to stand for 24 h at 15 degrees C. They were also exposed to dilutions of a primary-treated effluent for 30 days at 15 degrees C under semi-static conditions. Gill and gonad cylcooxygenase (COX) were then determined after the incubation period. The influence of various drugs found in municipal effluents on serotonin and dopamine synaptosome transport was determined in visceral ganglia. The results show that injections of ibuprofen reduced COX activity nearly 4-fold in gills and 1.4-fold in gonads. However, COX activity was induced in both tissues after 24 h in mussels injected with a municipal effluent extract and after 30 days in those exposed to dilutions of the effluent. Moreover, synaptosomal dopamine transport activity was increased by ibuprofen, aspirin, caffeine and estradiol-17beta (E2), and decreased by loperamide and carbamazepine, suggesting increased and decreased turnover rates of this catecholamine, respectively. Serotonin transport activity was much less affected, decreasing with high doses of loperamide and increasing with ibuprofen, but with less intensity than with dopamine. The results suggest that although ibuprofen can effectively reduce COX activity in gill and gonadal tissues, exposure to both the municipal effluent and its organic extract increased COX activities, indicating the absence of NSAID (non-steroidal anti-inflammatory drugs)-related effects. Besides their known estrogenic and serotonergic properties, municipal effluents appear to elicit a state similar to inflammation in freshwater mussels.     

Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year

Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.     

Neurochemical effects of benzodiazepine and morphine on freshwater mussels

The purpose of this study was to examine the neurochemical effects of morphine, diazepam, a common benzodiazepine, and an effluent concentrate on the endemic freshwater mussel Elliptio complanata. Mussels were exposed to the drugs and to the solid-phase concentrate of a municipal effluent and left to stand at 15 °C for 48 h. Neurochemical effects were determined by monitoring changes in dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) levels in the visceral mass (containing the nerve ganglia) of mussels. The activities of acetylcholinesterase (AChE), dopamine and serotonin-dependent adenylyl cyclase (ADC) were also determined in the mussels. Oxidative stress was determined by tracking changes in lipid peroxidation (LPO) in the mitochondrial and post-mitochondrial fractions. The results revealed that the drugs and the effluent extract were biologically active in mussels. Morphine reduced serotonin and increased dopamine in mussel tissues while reducing AChE activity and increasing GABA levels. This suggests the induction of a relaxation state in mussels. Diazepam also reduced serotonin levels but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, indicating the potential effect on opiate signaling. Diazepam increased glutamate levels slightly, but AChE remained stable. The increase in both dopamine ADC activity and glutamate concentrations was also associated with greater oxidative stress on the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity to morphine. In conclusion, the data warrant more studies on the analysis of opiates and benzodiazepines in municipal effluents to better address the potential environmental hazard of these neuroactive drug classes to aquatic organisms.     

Effects of municipal effluents on serotonin and dopamine levels in the freshwater mussel Elliptio complanata

Sex differentiation and gametogenesis represent critical steps in the reproductive process and are subject to hormonal control by serotonin, dopamine and steroids such as estradiol-17beta and testosterone. The purpose of this study sought to examine the endocrine-disrupting activity that a primary-treated municipal effluent might have on the metabolism of biogenic amine levels. First, serotonin receptors transfected in Chinese hamster ovary (CHO) cells were used to screen for the presence of serotonin receptor agonist or antagonist. Second, one group of Elliptio complanata mussels were exposed to single compounds likely to be found in municipal wastewaters and another group was exposed in situ to the municipal effluent plume for 90 days in experimental cages. Results showed that solid phase C-8 extracts of surface water downstream a municipal effluent could activate the transport of serotonin by receptors at a distance of at least 5 km from its outfall thereby indicating the presence of serotonin mimics in the effluent dispersion plume. Levels of serotonin and monoamine oxidase (MAO) activity in nerve ganglia of mussels exposed for 90 days to the municipal effluent were, respectively, reduced and increased at a distance 10-km downstream. Injections of estradiol-17beta and nonylphenol in mussels decreased the levels of serotonin and dopamine, but increased MAO activity in the gonad and nerve ganglia. Exposure to estrogenic chemicals present in municipal effluents may therefore alter the normal metabolism of serotonin and dopamine, both of which are involved in sexual differentiation in bivalves and fish. Chemicals acting through E2 receptor-mediated pathways and serotonin receptors are likely to cause the observed effects.     

Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata

Municipal effluents are an important source of estrogens to the aquatic environment. The purpose of this study was to examine the estrogenicity of municipal effluents to the indigenous freshwater mussel, Elliptio complanata. First, estradiol-binding sites in gonad homogenates were characterized to determine the binding affinity and specificity of estrogens. Mussels were exposed to increasing concentrations of a municipal effluent for 96 h at 15 degrees C. In another experiment, mussels were placed in cages and submerged for 62 days at 1.5 km upstream and 5 km downstream of a municipal effluent plume in the St. Lawrence River. Mussels were harvested for assessment of vitellogenin-like proteins in the hemolymph and determination of total lipid, carbohydrate and protein in the gonad. The presence of specific estrogen-binding sites was found in both male and female gonads. Binding of estradiol to cytosol proteins reached saturation, yielding a dissociation constant of 0.4 nM. Vitellogenin (Vg) levels increased significantly in both the hemolymph and the gonad after exposure to the effluent. Moreover, females appeared to be more sensitive than males to producing Vg. Mussels exposed in situ to contaminated surface waters had higher levels of Vg at the downstream site, again, females had higher levels of Vg than did males. On the other hand, lipid and sugar levels in male gonads were significantly increased at the downstream site. Moreover, mussels at the downstream site had decreased shell growth length and increased total and soft tissue weights. We conclude that municipal effluents contain bio-available xenoestrogens at levels sufficient to elicit effects in freshwater mussels.     

Neurotoxicological effects of a primary and ozonated treated wastewater on freshwater mussels exposed to an experimental flow-through system

The neurotoxic potential of a primary-treated and ozonated municipal effluent was examined using feral freshwater Elliptio complanata mussels. Specimens were exposed to increasing concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent before and after treatment with 10 mg/L of ozone in a mesocosm-type experiment for 30 days. A suite of biomarkers was used to assess the potential neurotoxic stress of the wastewaters on these benthic invertebrates: opiate binding sites, gamma-aminobutyric acid (GABA) metabolism, monoamines levels (serotonin, dopamine), monoamine oxidase, acetylcholinesterase and lipid peroxidation. Gametogenic activity was also determined by the gonado-somatic index and by vitellogenin-like proteins. The results show that the number of opiate binding sites increased slightly, especially after ozonation. GABA metabolism was generally reduced, suggesting higher glutamate stimulation than GABA dampening effects in mussel ganglia. This excitatory state was further confirmed by decreased acetylcholinesterase activity in gonadal tissues. The turnover of dopamine was enhanced with increased serotonin levels, but accompanied by reduced catabolism, as evidenced by decreased monoamine oxidase activity. Moreover, oxidative stress was increased, as determined by lipid peroxidation in the gonad (containing ganglia), which was significantly correlated with acetylcholinesterase activity and dopamine metabolism. The gonado-somatic index was significantly reduced with increased levels of vitellogenin-like proteins, again confirming the estrogenic action of these wastewaters. The data suggest that exposure to a primary-treated municipal effluent before and after ozonation leads to an excitotoxic syndrome implicating perturbations in GABA, dopamine and acetylcholine signaling. The increase in dopamine metabolism may be associated with the occurrence of opiate-like compounds (i.e. morphine) in the effluent. In general, ozonation reduced the severity of the responses, indicating that this disinfection strategy does not increase neurotoxicity to mussels.     

The effects of zinc oxide nanoparticles on the metallome in freshwater mussels

The use of zinc oxide nanoparticles (nanoZnO) as sunscreens has raised concerns about their safety and release in the aquatic environment through swimming activities and within municipally treated wastewaters. This study's purpose was to examine the effects of nanoZnO on the elemental composition (metallome) in exposed freshwater mussels, Elliptio complanata. Mussels were exposed for 21 days to an environmentally realistic (low) concentration (2 μg/L) of nanoZnO and zinc chloride. The mussels were also exposed to a physically and chemically treated municipal effluent (ME), both alone and in the presence of both forms of Zn. The metallome profile was characterized by the following 15 elements in gills, digestive gland and gonad tissues: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, V and Zn. The levels of metallothioneins (MT) and lipid peroxidation (LPO) in the digestive gland were also measured as biomarkers of toxic effects. The data revealed that exposure to nanoZnO increased the total levels of Zn, MT and LPO in the digestive gland. Discriminate function analysis revealed that the digestive gland responded the most to exposure to either nanoZnO or Zn^2 +. For nanoZnO, the observed changes in Al, As and Mo in the digestive gland offered the best discrimination from dissolved Zn^2 +. Co-exposure of nanoZnO with the ME changed the metallome profile closer to dissolved Zn^2 +, suggesting a common interaction site within the ME. This was observed in changes in Ni, Cu, Se and Zn in the digestive gland of exposed mussels. Canonical analysis of essential and non-essential elements revealed that exposure to nanoZnO increased the relationships between LPO and the sum of essential elements in the digestive gland. Conversely, exposure to dissolved Zn^2 + and the ME decreased the relationship between the sum of non-essential elements and LPO and MT. In conclusion, the use of a “metallomic” approach was used to discriminate changes following exposure to nanoZnO and dissolved Zn in freshwater mussels and provided insights into the interaction of forms of Zn in ME towards mussels.     

Effects of pharmaceutical products and municipal wastewaters on temperature-dependent mitochondrial electron transport activity in Elliptio complanata mussels

The advent of global warming has given rise to the issue on how temperature impacts the susceptibility of ectothermic organisms to pollution. The purpose of this study was to examine the effects of pharmaceutical products and domestic wastewaters on temperature-dependent mitochondrial electron transport activity in the freshwater mussel Elliptio complanata. Mitochondria from mussels were freshly prepared and exposed to increasing concentrations of various pharmaceutical products known to be found in municipal effluents for 30 min at 4, 12 and 20 degrees C. Electron transport activity as well as lipid peroxidation and DNA strand breaks were determined in the mitochondria. Next, mussels were placed in the aeration lagoons of two municipal wastewater treatment plants for 30 days. Mitochondrial electron transport (MET), temperature-dependent MET (MET(T)) and lipid peroxidation in gonad were then determined. The results show that all products were able to increase MET activity, but at two different ranges of threshold concentration. Certain pharmaceutical products (i.e., ibuprofen, cotinine, fluoxetine, coprostanol and trimethoprim) increased MET(T) at a lower threshold concentration than observed at 20 degrees C. Products of lesser potency in reducing lipid peroxidation were those that produced more DNA strand breaks in mitochondria. Both MET and MET(T) were significantly increased in mussels exposed to aeration lagoon effluents. Lipid peroxidation was also increased in the gonad and was significantly correlated with MET and MET(T) activities. The data indicate that pharmaceutical products and municipal effluents increase respiration rates in isolated mitochondria, such that interaction with temperature could enhance the susceptibility of mitochondrial energy production and oxidative stress in environments contaminated by domestic wastewater.     

Population dynamics of the green-lipped mussel, Perna canaliculus, at various spatial and temporal scales in northern New Zealand

Ecological processes that differentiate and maintain intertidal populations of mussels, Perna canaliculus, were studied within three sites at Ninety Mile Beach, northern New Zealand. At these three sites (Scott Point, The Bluff and Tonatona Beach), the dynamics of larval availability, primary and secondary settlement, recruitment and mortality rates were investigated at various spatial and temporal scales. (1) Mussel concentrations in seawater were variable with respect to study site and time of year, with highest abundances at the northernmost population (Scott Point) and lowest concentrations at the middle population (The Bluff). In seawater at all three sites, small mussels (< 0.25 mm in shell length) were more abundant in August 2000, while larger mussels (> 0.5 mm in shell length) were more abundant in March 2001. (2) Primary and secondary settlement patterns were investigated during short-term (daily) and long-term (monthly) settlement experiments, within quadrats that were cleared of all mussels in both the mussel bed and in adjacent algal band habitats. At all sites, primary settlement (< 0.5 mm in shell length) was high within the algal band habitat in August 1999, 2000 and 2001. Conversely, secondary settlement (> 2.0 mm in shell length) was high within cleared areas in the mussel beds in November-March 1999-2000 and 2000-2001. Abundance of mussels settling on artificial substrates placed in the intertidal did not differ greatly from comparable areas of natural substrates (bare rock or algae within cleared quadrats). (3) Recruitment and mortality rates were recorded during monthly surveys of the adult populations. Within three mussel size classes (< 24, 25-74 and > 75 mm in shell length), peak recruitment coincided with high mortality in August of the 2 years studied. However, the most dramatic turnover of the population was observed at Scott Point in both years, following a spawning event. In adjacent waters at Scott Point, large accumulations of drift algae covered (up to 100% cover) with juvenile mussels may deplete food supplies usually delivered to intertidal adult mussels, causing their demise. Mats of adult mussels were observed “peeling-off” from the rocky shore at this time of the year, making space available to the new recruits. Where nearshore algal accumulations were moderate to low, only moderate to low mussel turnovers were observed (e.g. Tonatona Beach and The Bluff).     

Tributyltin inhibits the oligomycin-sensitive Mg-ATPase activity in Mytilus galloprovincialis digestive gland mitochondria

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filter-feeding molluscs. TBT is known to act as a membrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM (203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μM TBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure.     

Protective role of Mangifera indica, Cucumis melo and Citrullus vulgaris peel extracts in chemically induced hypothyroidism

An investigation was made to evaluate the pharmacological importance of fruit peel extracts of Mangifera indica (MI), Citrullus vulgaris (CV) and Cucumis melo (CM) with respect to the possible regulation of tissue lipid peroxidation (LPO), thyroid dysfunctions, lipid and glucose metabolism. Pre-standardized doses (200 mg/kg of MI and 100 mg/kg both of CV and CM), based on the maximum inhibition in hepatic LPO, were administered to Wistar albino male rats for 10 consecutive days and the changes in tissue (heart, liver and kidney) LPO and in the concentrations of serum triiodothyronine (T₃), thyroxin (T₄), insulin, glucose, α-amylase and different lipids were examined. Administration of three test peel extracts significantly increased both the thyroid hormones (T₃ and T₄) with a concomitant decrease in tissue LPO, suggesting their thyroid stimulatory and antiperoxidative role. This thyroid stimulatory nature was also exhibited in propylthiouracil (PTU) induced hypothyroid animals. However, only minor influence was observed in serum lipid profile in which CM reduced the concentrations of total cholesterol and low-density lipoprotein-cholesterol (LDL-C), while CV decreased triglycerides and very low-density lipoprotein-cholesterol (VLDL-C). When the combined effects of either two (MI + CV) or three (MI + CV + CM) peel extracts were evaluated in euthyroid animals, serum T₃ concentration was increased in response to MI + CV and MI + CV + CM treatments, while T₄ level was elevated by the combinations of first two peels only. Interestingly, both the categories of combinations increased T₄ levels, but not T₃ in PTU treated hypothyroid animals. Moreover, a parallel increase in hepatic and renal LPO was observed in these animals, suggesting their unsafe nature in combination. In conclusion the three test peel extracts appear to be stimulatory to thyroid functions and inhibitory to tissue LPO but only when treated individually.     

Serotonergic mechanisms mediating spawning and oocyte maturation in the zebra mussel,Dreissena polymorpha

Summary

The zebra mussel, Dreissena polymorpha, is a freshwater biofouling bivalve unintentionally introduced in the 1980s into North America from Europe. Oocyte maturation (germinal vesicle breakdown, GVBD) and spawning of the zebra mussel can be triggered with serotonin (5-hydroxytryptamine, 5-HT). In pharmacological experiments to characterize the receptor mediating spawning, the serotonin receptor agonists 8-OH-DPAT, TFMPP, and 1-(1-naphthyl)piperazine were effective at stimulating spawning; whereas, 2-methylserotonin and alpha-methylserotonin had no effect. In experiments with antagonists of serotonin receptors ketanserin and propranolol had no effect; mianserin, NAN-190, and cyproheptadine had partial inhibitory effects; and methiothepin was a very effective antagonist. Metergoline had mixed agonist/antagonist properties. Ergotamine was the most effective activator of spawning in females. Compared to serotonergic receptors in other organisms, the receptors that activate spawning in zebra mussels resemble 5HTlym, 5HTdro2 and human 5HT1Dβ, which are receptors that may act both by inhibiting adenylyl cyclase and by activating phospholipase C. In zebra mussels, 5-HT and 8-OH-DPAT activate GVBD in gonad fragments, a process also initiated by manual dissection of gonad fragments. GVBD can be inhibited by pre-treatment of ovaries with forskolin and theophylline, suggesting an inhibitory role for cyclic AMP. The Ca²⁺ ionophore A23187 can trigger GVBD and polar body formation. Thus, oocyte maturation in zebra mussels may be initiated via serotonergic receptors simultaneously inhibiting adenylyl cyclase and activating Ca²⁺ mechanisms.     

Effect of air exposure on lysosomal tissues of Mytilus edulis L. from natural intertidal wild beds and submerged culture ropes

Blue mussels collected from suspended culture ropes and from three natural intertidal wild beds from different areas of the German Bight were tested for their ability to cope with hypoxic conditions. During the experiment mussels were exposed to air from 0 to 72 h. Mussels from all sampling sites displayed high tolerance to aerial exposure with moderate levels of mortality after 12 to 48 h of exposure. Lysosomal membrane stability (LMS), a biomarker of general stress, changed notably between minimum values after 12 h and maximum values after 24 h of aerial exposure in intertidal mussels. In contrast, labilization times of mussels from the hanging culture increased continuously up to 48 h of exposure. Intertidal mussels from the island of Heligoland exhibited significantly decreased membrane stability after 72 h of air exposure, correlating to higher mortality rates. Intertidal mussels, although adapted to daily aerial exposure in their natural environment, showed a similar pattern of mortality and lower LMS values during the experiment than mussels from the suspended culture site. The increase of LMS values of mussels under hypoxic conditions at the beginning of the experiment at all sites was tested for the influence of macro-autophagic processes using immune labelling techniques. With this approach it could be demonstrated that high LMS values significantly correlate with low autophagic activity. However, hypoxic conditions do not enhance autophagic processes during the early periods of aerial exposure. Only at the end of the experiment, high values for autophagy were measured in mussels from an intertidal site accompanied with high mortalities. The results indicate that autophagic processes are not involved in the early adaptive processes that enable the mussel to cope with periods of aerial exposure.     

The effect of photoperiod on the reproductive development of the northern bay scallop, Argopecten irradians irradians

One-year-old bay scallops, Argopecten irradians irradians (58 ± 2 mm, 22 ± 1 g live weight) were exposed to four replicated photoperiod treatments (24D, 8L:16D, 16L:8D, and 24L where D = dark hours, L = light hours) in order to measure the effect on gonad weight and maturation during the conditioning process. Results indicated that day-lengths of more than 8 h are necessary to promote gonad maturation in bay scallops. After 6 wk, the mean gonad weight for scallops in the 16-h and 24-h light regimes was similar at 0.6 ± 0.1 g dry weight compared to a mean of 0.2 ± 0.1 g dry weight for those in the 8-h and 0-h light regimes. Histological assessment indicated significantly more follicular tissue development in both the male and female portion of the gonad in the two longer photoperiod treatments. Overall, gamete maturity was highest for the scallops in the 16-h light regime; the incidence of mature eggs was 50% compared to 35% in the 24-h light regime, 20% in the 8-h light regime and 10% in the 0-h light regime. Assessment of feeding rates indicated no significant difference in algal cell consumption among treatments. Total dry tissue weight doubled over the 6-wk conditioning trial with no significant differences among treatments. One-year-old bay scallops appear to be non-responsive to conditions suitable for gonad maturation (i.e. appropriate temperature and food levels) unless they receive more than 8 h of light exposure. This finding has important implications for northern hatcheries which typically condition broodstock indoors during the early spring.     

Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure

This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0–48 µg L^− 1) from embryonic to juvenile stages for 80 days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.     

Intermolecular association provides specific optical and NMR signatures for serotonin at intravesicular concentrations

Neurotransmitter vesicles contain biomolecules at extraordinarily high concentrations (hundreds of millimoles/liter). Such concentrations can drive intermolecular associations, which may affect vesicular osmolarity and neuronal signaling. Here we investigate whether aqueous serotonin (a monoamine neurotransmitter) forms oligomers at intravesicular concentrations and whether these oligomers have specific spectroscopic signatures that can potentially be used for monitoring neuronal storage and release. We report that, as serotonin concentration is increased from 60 μM to 600 mM, the normalized fluorescence spectrum of serotonin displays a growing long-wavelength tail, with an isoemissive point at 376 nm. The fluorescence decay is monoexponential with a lifetime of 4 ns at low concentrations but is multiexponential with an average lifetime of 0.41 ns at 600 mM. A 600 mM serotonin solution has 30% less osmolarity than expected for monomeric serotonin, indicating oligomer formation. The proton NMR chemical shifts move upfield by as much as 0.3 ppm at 600 mM compared to those at 10 mM, indicating a stacking of the serotonin indole moieties. However, no intermolecular crosspeak is evident in the two-dimensional NMR rotating frame Overhauser effect spectroscopy spectrum even at 600 mM, suggesting that oligomeric structures are possibly weakly coupled. The appearance of a single peak for each proton suggests that the rate of interconversion between the monomeric and the oligomeric structures is faster than 240 Hz. A stopped-flow kinetic experiment also confirms that the rate of dissociation is faster than 100 ms. We conclude that serotonin forms oligomers at intravesicular concentrations but becomes monomeric quickly on dilution. NMR signatures of the oligomers provide potential contrast agents for monitoring the activity of serotonergic neurons in vivo.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Discovery of N-methyl-1-(1-phenylcyclohexyl)ethanamine, a novel triple serotonin, norepinephrine and dopamine reuptake inhibitor

Novel chiral cyclohexylaryl amines were developed with potent reuptake inhibition against the serotonin, norepinephrine and dopamine transporters and activity at 10 and 30 mpk PO in the mouse tail suspension test. Prototype compound 31 (SERT, NET, DAT IC₅₀ ? 1, 21, 28 nM) was highly brain penetrant, had minimal CYP and hERG inhibition, and represents a previously undisclosed architecture with potential for treatment of major depressive disorder.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.