The role of cysteine conjugation in the detoxification of microcystin-LR in liver of bighead carp (Aristichthys nobilis): a field and laboratory study

The role of glutathione (GSH) and cysteine (Cys) conjugates in the detoxification of microcystin-LR (MC-LR) in bighead carp (Aristichthys nobilis) was examined under laboratory and field conditions. Wild individuals of bighead carp were collected from 5 eutrophic lakes along the Yangtze River, while in laboratory experiment, bighead carp were injected intraperitoneally with 500 μg purified MC-LR/kg body weight (bw). Contents of MC-LR and its glutathione (MC-LR-GSH) and cysteine conjugates (MC-LR-Cys) in the liver of bighead carp were determined by liquid chromatography electrospray ionization mass spectrum (LC–ESI–MS). In laboratory experiment, low concentrations of MC-LR-GSH (mean: 0.042 μg/g dry weight (DW)) were always detectable, and the mean ratio of MC-LR-Cys to MC-LR-GSH was 6.55. While, in field study, relatively high MC-LR-Cys concentration (mean: 0.22 μg/g DW) was detected, whereas MC-LR-GSH was occasionally detectable, and the average ratio of MC-LR-Cys to MC-LR-GSH was as high as 71.49. A positive correlation was found between MC-LR-Cys concentration in the liver of bighead carp and MC-LR content in seston from the five lakes (r = 0.85). These results suggest that MC-LR-Cys might be much more important than MC-LR-GSH in the detoxification of MC-LR in fish liver, and that cysteine conjugation of MC-LR might be a physiological mechanism for the phytoplanktivorous bighead carp to counteract toxic cyanobacteria.     

Time-dependent oxidative stress and histopathological changes in <Emphasis Type="Italic">Cyprinus carpio</Emphasis> L. exposed to microcystin-LR

Microcystins (MCs) are produced by cyanobacteria in aquatic environments and are a potential risk to aquatic organisms. Increasing evidence suggests that oxidative stress may play an important role in the toxicity mechanism of MCs on fish, but most studies were based on relatively high concentrations. In this study, the effect of time-dependent oxidative stress in livers of Cyprinus carpio L. (C. carpio) exposed to 10 μg l⁻¹ of microcystin-LR (MC-LR) for 0–14 days was investigated. MC-LR induced histopathological changes in liver and gills were also assessed after 14 days exposure. Electron paramagnetic resonance (EPR) spectrum was used to directly investigate the reactive oxygen species (ROS) in fish liver and results showed that hydroxyl radical (^∙OH) was significantly induced at 0.5 day and then tended to decline with an increase of exposure period. As a response of antioxidant, catalase (CAT) activity increased slightly at first and then decreased with exposure period. A pronounced promotion of glutathione-S-transferase (GST) indicated that the conjugation reaction of MC-LR and GSH occurred. A time-dependent decrease of reduced glutathione (GSH) with an increase of oxidized glutathione (GSSG) level suggested GSH was involved in detoxification of MC-LR in the liver. Oxidative damage was evidenced by the significant increase of malondialdehyde (MDA) level at 2–6 days. After 14 days exposure, a series of pathological changes, like partially dissolved parenchymal architecture, vacuolar degeneration, necrosis, hemorrhage and slight inflammatory cells infiltration in fish liver tissues could be observed. Scanning electron microscopic (SEM) studies showed that dissolved MC-LR could also result in pathological changes like partial broken epithelial cells, deformed taste buds and loose gill filament and lamella in gill tissues. These results suggest that although a restoring response occurred, C. carpio could still be adversely affected by MC-LR at 10 μg l⁻¹.     

Effects of an anionic surfactant (FFD-6) on the energy and information flow between a primary producer (<Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>) and a consumer (<Emphasis Type="Italic">Daphnia magna</Emphasis>)

The effects of a commercially available anionic surfactant solution (FFD-6) on growth and morphology of a common green alga (Scenedesmus obliquus) and on survival and clearance rates of the water flea Daphnia magna were studied. The surfactant-solution elicited a morphological response (formation of colonies) in Scenedesmus at concentrations of 10–100 μl l⁻¹ that were far below the No Observed Effect Concentration (NOEC) value of 1,000 μl l⁻¹ for growth inhibition. The NOEC-value of FFD-6 for colony-induction was 3 μl l⁻¹. Daphnia survival was strongly affected by FFD-6, yielding LC_50–24h and LC_50–48h of 148 and 26 μl l⁻¹, respectively. In addition, clearance rates of Daphnia feeding on unicellular Scenedesmus were inhibited by FFD-6, yielding a 50% inhibition (EC_50–1.5h) at 5.2 μl l⁻¹ with a NOEC of 0.5 μl l⁻¹. When Daphnia were offered FFD-6-induced food in which eight-celled colonies (43 × 29 μm) were most abundant, clearance rates (~0.14 ml ind.⁻¹ h⁻¹) were only 25% the rates of animals that were offered non-induced unicellular (15 × 5 μm) Scenedesmus (~0.56 ml ind.⁻¹ h⁻¹). As FFD-6 concentrations in the treated food used in the experiments were far below the NOEC for clearance rate inhibition, it is concluded that the feeding rate depression was caused by the altered morphology of the Scenedesmus moving them out of the feeding window of the daphnids. The surfactant evoked a response in Scenedesmus that is similar to the natural chemically induced defensive reaction against grazers and could disrupt the natural information conveyance between these plankton organisms.     

Assessment of estrogenic contamination and biological effects in Lake Taihu

Lake Taihu is the third largest freshwater lake in China and is contaminated with xenoestrogens associated with high population density, intensive livestock and aquatic breeding activities. A field study in Lake Taihu was conducted using the goldfish (Carassius auratus) as an indicator organism. Several biological markers were selected to assess the extent of estrogenic contamination. Changes in serum vitellogenin (VTG), and gill 7-Ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST) and reduced glutathione (GSH) were measured in caged juvenile goldfish for 28 days in seven locations in northern Lake Taihu. Bioassay showed VTG increased 0.64–2.42 folds over time in goldfish collected from five stations and GSH decreased in samples from all seven stations after 7 days of exposure. EROD levels increased continually in fish collected at all the seven stations and the highest concentrations occurred at day 21. GST activity increased significantly at 7 days. The concentration of the target estrogens estrone (E₁), 17β-estradiol (E₂), ethinylestradiol (EE₂), octylphenol (OP), diethylstilbestrol (DES), nonylphenol (NP) and bisphenol A (BPA) were determined in lake water at the sampling stations. Each individual estrogen concentration measured was multiplied by its relative potency to gain the estrdiol equivalent (EEQ). There was an obvious correlation between the concentration of VTG and the total EEQ for all seven locations (P < 0.001). The biomarker VTG, EROD, GST and GSH assays and chemical analysis might be used to illustrate the potential risk in Lake Taihu.     

Suitability of <Emphasis Type="Italic">Daphnia similis</Emphasis> as an alternative organism in ecotoxicological tests: implications for metal toxicity

The acute toxicity of metals to Daphnia similis was determined and compared to other daphnid species to evaluate the suitability of this organism in ecotoxicology bioassays. To verify the performance D. similis in toxicity tests, we also investigated the effect of Pseudokirchneriella subcapitata at 1 × 10⁵ and 1 × 10⁶ cells ml⁻¹ on Cd and Cr acute toxicity to the cladoceran. Daphnid neonates were exposed to a range of chromium and cadmium concentrations in the absence and presence of the algal cells. Metal speciation calculations using MINEQL⁺ showed that total dissolved metal concentrations in zooplankton culture corresponded to 96.2% free Cd and 100% free Cr concentrations. Initial total dissolved metal concentrations were used for 48 h-LC₅₀ determination. LC₅₀ for D. similis was 5.15 × 10⁻⁷ mol l⁻¹ dissolved Cd without algal cells, whereas with 1 × 10⁵ cells ml⁻¹, it was significantly higher (7.15 × 10⁻⁷ mol l⁻¹ dissolved Cd). For Cr, the 48 h-LC₅₀ value of 9.17 × 10⁻⁷ mol l⁻¹ obtained for the cladoceran in tests with 1 × 10⁶ cells ml⁻¹ of P. subcapitata was also significantly higher than that obtained in tests without algal cells (5.28 × 10⁻⁷ mol l⁻¹ dissolved Cr). The presence of algal cells reduced the toxicity of metals to D. similis, as observed in other studies that investigated the effects of food on metal toxicity to standard cladocerans. Comparing our results to those of literature, we observed that D. similis is as sensitive to metals as other standardized Daphnia species and may serve as a potential test species in ecotoxicological evaluations.     

Dissolved organic carbon reduces uranium toxicity to the unicellular eukaryote <Emphasis Type="Italic">Euglena gracilis</Emphasis>

The influence of dissolved organic carbon (DOC), in the form of Suwannee River fulvic acid (SRFA), on uranium (U) toxicity to the unicellular eukaryote, Euglena gracilis (Z strain), was investigated at pH 6. In a background medium without SRFA, exposure of E. gracilis to 57 μg L⁻¹ U resulted in a 50% reduction in growth (IC₅₀). The addition of 20 mg L⁻¹ DOC (as SRFA), reduced U toxicity 4 to 5-fold (IC₅₀ increased to 254 μg L⁻¹ U). This reduction in toxicity was also evident at more sensitive effect levels with a 10% reduction in growth (IC₁₀) occurring at 5 μg L⁻¹ U in the background medium and at 17 μg L⁻¹ U in the SRFA medium, respectively. This amelioration of toxicity with the addition of SRFA was linked to a decrease in the bioavailability of U, with geochemical speciation modelling predicting 84% of U would be complexed by SRFA. The decrease in bioavailability of U in the presence of SRFA was also evident from the 11–14 fold reduction in the cellular concentration of U compared to that of E. gracilis in the background medium. Stepwise multiple linear regression analyses indicated that UO₂ ²⁺ alone explained 51% of the variation in measured U toxicity to E. gracilis. Preliminary U exposures to E. gracilis in the presence of a reactive oxygen species probe, suggest exposure to ≥60 μg L⁻¹ U may induce oxidative stress, but this endpoint was not considered to be a sensitive biological indicator.     

Tolerance and accumulation of lead by species of <Emphasis Type="Italic">Iris</Emphasis> L.

Seedling development, accumulation and distribution of lead (Pb) in Iris lactea var. chinensis (Fisch.) Koidz. and I. tectorum Maxim. were studied using plants grown in sand culture and exposed to 0–10 mmol l⁻¹ concentrations of Pb supplied as Pb(NO₃)₂ for 28 days. A significant reduction in dry weight (dw) of shoots and roots of I. lactea var. chinensis was observed at 6 and 10 mmol l⁻¹, respectively, and a significant reduction in dw of shoots and roots of I. tectorum was observed at 6 mmol l⁻¹. Concentration of Pb in the shoots and roots of I. lacteal var. chinensis exposed to 4 mmol l⁻¹ Pb reached 1,109 μg g⁻¹ and 2,408 μg g⁻¹ dw, respectively. The index of tolerance (IT) of I. lactea var. chinensis among 0–8 mmol l⁻¹ Pb treatments were not significantly different, while those of I. tectorum at 6 mmol l⁻¹ Pb were significantly decreased. The results indicated that I. lactea var. chinensis was more tolerant to Pb than I. tectorum. Sub-cellular localization of Pb in root cells was evaluated using transmission electron microscopy (TEM) and Pb deposits were found along the plasma membrane of some root tip cells of I. lactea var. chinensis treated at 10 mmol l⁻¹ Pb. Deposits of Pd were also observed along the surface, in the root tip cell wall and in the cytoplasm of a few malformed cells of I. tectorum exposed at 10 mmol l⁻¹ Pb treatment. One possible mechanism to explain these observations may be that most cells can maintain normal activities in the plant by sacrificing a small number of cells that accumulate a large amount Pb and show toxicity. Future studies should be designed to test this hypothesis.     

Acute and chronic effects of sodium and potassium on the tropical freshwater cladoceran <Emphasis Type="Italic">Pseudosida ramosa</Emphasis>

In this study, the toxicities of sodium and potassium to the tropical freshwater cladoceran Pseudosida ramosa were assessed. Acute toxicity tests on this species showed that the 48-h LC₅₀ of Na⁺ was 556 mg l⁻¹, while that of K⁺ was 17.7 mg l⁻¹. Long-term exposure of female P. ramosa to sodium reduced the total number of survivors from 10 to 6 at a concentration of 249 mg l⁻¹, 21-day fecundity from 20.4 to 14.3 eggs female⁻¹ at concentrations ranging from 72 to 249 mg l⁻¹, 21-day fertility from 20.1 to 6.5 neonates female⁻¹ at concentrations ranging from 25 to 249 mg l⁻¹. Furthermore, fecundity of each brood from the second to the fifth was significantly lower at 249 mg l⁻¹ and fertility of each brood from the first to the fifth at concentrations ranging from 25 to 249 mg l⁻¹. A significant decrease in fertility was associated with an increase in the number of aborted eggs. Long-term exposure to potassium decreased the 21-day fecundity of P. ramosa from 14.2 to 10.8 eggs female⁻¹ at a concentration of 11 mg l⁻¹ and fertility (fourth brood only) at 6.2 and 11 mg l⁻¹. Tropical reservoirs located near areas where the soil is overloaded with fertilizers and ferti-irrigation with vinasse already show concentrations of Na⁺ and K⁺ very close to those producing sub-lethal long-term effects on P. ramosa. A possible consequence is that organisms of the aquatic biota cannot adapt and freshwater taxa may become locally extinct, transferring dominance to salt-tolerant taxa.     

Effect of nitroxyalkyl derivatives of fullerenylproline on the activity of CA<Superscript>3+</Superscript>-ATPase of sarcoplasmic reticulum

The effect of nitroxyalkyl derivatives of fullerenylproline methyl ester on the enzymatic activity of Ca²⁺-ATPase of sarcoplasmic reticulum (SR) has been studied. It is shown that hybrid derivatives of C₆₀ fullerene are capable of inhibiting the activity of Ca²⁺-ATPase of SR. The mononitrate inhibits the hydrolytic activity of the enzyme with K _i = 1.92 × 10⁻⁶ M; active Ca²⁺ transport, with K _i = 3.79 × 10⁻⁶ M. The dinitrate inhibits ATP hydrolysis with K _i = 2.38 × 10⁻⁸ M; Ca²⁺ transport, with K _i  = 3.08 × 10⁻⁸ M. Fullerenylproline methyl ester does not affect the enzymatic activity of Ca²⁺-ATPase. Based on these data it is possible to predict the possible fields of application for hybrid fullerene derivatives as potential drugs.     

Toxicity of lead, cadmium and mercury on embryogenesis, survival, growth and metamorphosis of Meretrix meretrix larvae

In order to assess the toxicity of heavy metals on the early development of Meretrix meretrix, the effects of mercury (Hg), cadmium (Cd) and lead (Pb) on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC₅₀ for embryogenesis was 5.4 μg l⁻¹ for Hg, 1014 μg l⁻¹ for Cd and 297 μg l⁻¹ for Pb, respectively. The 96 h LC₅₀ for D-shaped larvae was 14.0 μg l⁻¹ for Hg, 68 μg l⁻¹ for Cd and 353 μg l⁻¹ for Pb, respectively. Growth was significantly retarded at 18.5 μg l⁻¹ (0.1 μM) for Hg, 104 μg l⁻¹ (1 μM) for Cd and 197 μg l⁻¹ (1 μM) for Pb, respectively. The EC₅₀ for metamorphosis, similar to 48 h LC₅₀, was higher than 96 h LC₅₀. Our results indicate that the early development of M. meretrix is highly sensitive to heavy metals and can be used as a test organism for ecotoxicology bioassays in temperate and subtropical regions.     

Assessment of Blood–Brain Barrier Permeability Using the <Emphasis Type="BoldItalic">In Situ</Emphasis> Mouse Brain Perfusion Technique

Purpose  To assess the blood–brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+) and mdr1a(−/−) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and P-gp-mediated efflux on the rate of brain uptake. Methods  The BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil) to slowly-equilibrating (fexofenadine), was determined and compared. Results  There was a sigmoidal relationship in mdr1a(−/−) mice between the log-PS and clogD_7.4 in the range of 0–5. The brain uptake clearance was a function of both permeability and blood flow rate. The brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium approximately 4,000-fold faster than fexofenadine, based on the magnitude of PS×fu,brain. Conclusions   In situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity, ionization state, molecular weight and polar surface area are all important determinants for brain penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is determined by a multiplicity of pharmacokinetic factors.     

Feedback modeling of non-esterified fatty acids in rats after nicotinic acid infusions

A feedback model was developed to describe the tolerance and oscillatory rebound seen in non-esterified fatty acid (NEFA) plasma concentrations following intravenous infusions of nicotinic acid (NiAc) to male Sprague-Dawley rats. NiAc was administered as an intravenous infusion over 30 min (0, 1, 5 or 20 μmol kg⁻¹ of body weight) or over 300 min (0, 5, 10 or 51 μmol kg⁻¹ of body weight), to healthy rats (n = 63), and serial arterial blood samples were taken for measurement of NiAc and NEFA plasma concentrations. Data were analyzed using nonlinear mixed effects modeling (NONMEM). The disposition of NiAc was described by a two-compartment model with endogenous turnover rate and two parallel capacity-limited elimination processes. The plasma concentration of NiAc was driving NEFA (R) turnover via an inhibitory drug-mechanism function acting on the formation of NEFA. The NEFA turnover was described by a feedback model with a moderator distributed over a series of transit compartments, where the first compartment (M ₁) inhibited the formation of R and the last compartment (M _N ) stimulated the loss of R. All processes regulating plasma NEFA concentrations were assumed to be captured by the moderator function. The potency, IC ₅₀, of NiAc was 45 nmol L⁻¹, the fractional turnover rate k _out was 0.41 L mmol⁻¹ min⁻¹ and the turnover rate of moderator k _tol was 0.027 min⁻¹. A lower physiological limit of NEFA was modeled as a NiAc-independent release (k _cap ) of NEFA into plasma and was estimated to 0.032 mmol L⁻¹ min⁻¹. This model can be used to provide information about factors that determine the time-course of NEFA response following different modes, rates and routes of administration of NiAc. The proposed model may also serve as a preclinical tool for analyzing and simulating drug-induced changes in plasma NEFA concentrations after treatment with NiAc or NiAc analogues.     

Hypoxia-induced oxidative DNA damage links with higher level biological effects including specific growth rate in common carp, Cyprinus carpio L

Both hypoxia and hyperoxia, albeit in different magnitude, are known stressors in the aquatic environment. Adopting an integrated approach, mirror carp (Cyprinus carpio L.), were exposed chronically (i.e. 30 days) to hypoxic (1.8 ± 1.1 mg O₂ l⁻¹) and hyperoxic (12.3 ± 0.5 mg O₂ l⁻¹) conditions and resultant biological responses or biomarkers were compared between these two treatments as well as with fish held under normoxic conditions (7.1 ± 1.04 mg O₂ l⁻¹). The biomarkers determined included the activities of glutathione peroxidase (GPx), measurement of oxidative DNA damage (using modified Comet assay employing bacterial enzymes: Fpg and Endo-III), haematological parameters, histopathological and ultrastructural examination of liver and gills. Specific growth rate (SGR) of the fish, as an important ecotoxicological parameter was also determined over the exposure period. The study suggested that while the levels of hepatic GPx were unaffected, there was a significant difference in activity in the blood plasma under different exposure conditions; the hyperoxic group showed increased GPx activity by approximately 37% compared to normoxic group and the hypoxic group showed a decrease by approximately 38% than the normoxic group. Interestingly, oxidative DNA damage was significantly higher in both hypoxic and hyperoxic by approximately 25% compared to normoxic conditions, Fpg showing enhanced level of damage compared to the Endo-III treatment (P < 0.001). The haematological parameters showed enhanced values under hypoxic conditions. Transmission electron microscopic (TEM) studies revealed damage to liver and gill tissues for both the treatments. Interestingly, SGR of fish was significantly lowered in hypoxic by approx. 30% compared to normoxic condition and this was found to be correlated with DNA damage (R = −0.82; P = 0.02). Taken together, these results indicate that prolonged exposure to both hypoxic and hyperoxic conditions induce oxidative stress responses at both DNA and tissue levels, and hypoxia can result in compensatory changes in haematological and growth parameters which could influence Darwinian fitness of the biota with wider ecological implications.     

Assessing the toxicity of ingested Taihu Lake water on mice via hepatic histopathology and matrix metalloproteinase expression

Water from Taihu Lake (China) is used as a drinking source. The frequency of contamination in Taihu Lake has increased over the past decade and the bloom-forming cyanobacteria are the dominant species during eutrophication. Cyanobacteria can produce various harmful secondary substances including microcystins capable of endangering human health and ecological safety. This study investigated toxicity of ingested Taihu Lake water on mice via hepatic histopathology and matrix metalloproteinase (MMP) expression. Water was sampled from four Taihu Lake locations, Meiliang Bay 2 group (M2), Meiliang Bay 1 group (M1), Lake Center (H) and Xukou Bay (X), along a gradient of decreasing degree of eutrophication. The experimental design consists of five groups of male mice (Mus musculus, ICR): one control and four groups ingesting water from the four sampling sites for 90 days. Compared to control, M1 and M2 mice showed hepatic histopathological changes including swollen, vacuolar degeneration or inflammatory. Immunohistochemical staining demonstrated a higher expression of MMP-2 proteins in M2 group and a lower expression of MMP-9 in M1. Enzyme-linked immunosorbent assay indicated that MMP-9 concentration was significantly reduced from 0.55 to 0.28 ng/g liver weight in M2 (p < 0.05). Real time PCR revealed a down-regulation of MMP-9 mRNA by 2.2 fold in M1 and an up-regulation of MMP-2 mRNA by 1.73 fold in H. Using this mouse model as a gauge of water toxicity, our results revealed that potential health risks induced by Taihu Lake water might arise from the use of this source water by local resident.     

Evaluation of Some Pharmacological Activities of ethanol extracts of seeds, pericarp and leaves of Eugenia Jamolana in Rats

The present study investigated the effect of ethanol extracts of seeds, pericarp and leaves of Eugenia Jamolana (E. Jamolana) on inflammation, gastric ulcer, anti-oxidants and hepatoprotective in rats. The acute inflammation was induced by intra-plantar injection of carrageenan (100 μl of 1 %) in the rat hind paw. Gastric ulcer was evoked by indomethacin (25 mg/kg) oral administration. Liver damage was induced by given CCL₄ (2.5 ml/kg) orally. The median lethal (LD₅₀) of the ethanol extract of both seeds and pericarp were determined and revealed that the investigated extracts of seeds and pericarp were non toxic up to 5g/kg. The anti-inflammatory results showed that the oral administration of ethanol extract of E. Jamolana seeds (250,500 mg/kg) showed significant inhibition of oedema formation in dose-dependent manner by −27.86, −41.23, −44.73, −51.78 % and by −63.16, −37.77, −47.04, −55.36 % at 1, 2, 3 and 4 h at 1, 2, 3 and 4 h, respectively. While the pericarp given at dose (500 mg/kg) exhibited significant inhibition of the oedema formation by −34.64, −21,8, 19.23 and −33.47 % at 1, 2, 3 and 4 h, respectively post carrageenan injection as compared with saline control group. E. Jamolana leaves fraction 1 given orally at dose of 25 mg/kg, induced non significant change on oedema, while the oedema response was significantly inhibited by −25.14, −33.4, −20.57 and −26.46 % at 1, 2, 3 and 4 h, respectively in group of rats that received leaves fraction 2 at the same dose. Rats were given leaves fraction 3 extract showed inhibition of oedema formation by −4.48 % at 1^st h post- carrageenan injection, while at 2^nd, 3^rd and 4^th h showed non significant change on oedema formation. The acute gastric mucosal lesions was significantly reduced by given ethanol extract of E. Jamolana seeds, pericarp (250, 500 mg/kg) and leaves fractions 1, 2 and 3 (25 mg/kg) respectively in dose dependent manner, as compared with indomethacin treated group (control group). All tested extracts showed significant reduction in elevated serum ALT, AST and ALP levels as compared with CCl ₄ treated group. The ethanol extract of E. Jamolana seeds, pericarp and leaves fractions 1, 2, 3 showed significant elevation of blood GSH level and significant reduction in elevated plasma lipid peroxides (MDA) as compared with CCl₄ treated group. In conclusion we can see that the ethanol extracts of E. Jamolana of seeds, pericarp and leaves fractions showed anti-inflammatory, anti-ulcer, hepatoprotective and anti-oxidants activity. Received 15 June 2008; accepted 11 November 2008     

Change in life cycle parameters and feeding rate of <Emphasis Type="Italic">Ceriodaphnia silvestrii</Emphasis> Daday (Crustacea,Cladocera) exposure to dietary copper

Changes in life cycle parameters (survival, growth, reproduction) and feeding rate of the tropical cladoceran Ceriodaphnia silvestrii as affected by Cu contaminated algae Pseudokirchneriella subcapitata were investigated. The dietary copper exposure ranged from 3 × 10⁻¹⁵ to 68 × 10⁻¹⁵ g Cu algal cell⁻¹. Low waterborne copper exposure (around 10⁻¹⁰ mol l⁻¹ free Cu²⁺ ions) was kept in the experiments. The results show an increasing toxic effect on C. silvestrii with copper increase in algal cells; at the highest copper exposure, all life cycle parameters were significantly affected. A concentration of 38 × 10⁻¹⁵ g Cu algal cell⁻¹ reduced egg hatching percentile and the number of neonates produced per female, but did not cause any statistically significant effect on animals survival nor to the number of eggs produced per female. The following sequence of events was observed from the lowest to the highest copper contamination: reproduction, feeding rate, body length and, at last, survival was affected. We conclude that algal cells are an important route of copper exposure and toxicity to cladocerans.     

Actions of kurtoxin on tetrodotoxin-sensitive voltage-gated Na<Superscript>+</Superscript> currents,Na<Subscript>V</Subscript>1.6, in murine vas deferens myocytes

Kurtoxin is described as a selective inhibitor of Ca_V3.1. Using patch-clamp techniques, the modulatory effects of kurtoxin on tetrodotoxin-sensitive voltage-gated Na⁺ currents (I _Na) recorded from mouse vas deferens myocytes were investigated. Kurtoxin increased the peak amplitude of I _Na between −40 and −30 mV, whilst inhibited the peak amplitude at more positive potentials than −10 mV, thereby demonstrating a dual action on the peak amplitude of I _Na. The time to reach the peak amplitude of I _Na became significantly longer in the presence of kurtoxin in comparison with that of the controls. Kurtoxin also slowed the deactivation of I _Na at more positive membrane potentials than −30 mV. Kurtoxin enhanced the total amount of electrical charge of I _Na in a concentration-dependent manner. These results suggest that kurtoxin is a modulator of Na_V1.6 in native freshly dispersed smooth muscle cells from mouse vas deferens as well as its action on Ca_V3.1.     

Evaluation of genotoxicity and effects on reproduction of nonylphenol in <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> (Pisces: cichlidae)

Nonylphenol ethoxylate (NPE) is widely used as a component of detergents, paints, pesticides, and many other products. In the aquatic environment NPE breakdown to 4-nonylphenol (NP), which is more stable and persistent. NP is estrogenic in fish, avian, and mammals and is described as an environmental pollutant with endocrine disruptor characteristics. The genotoxicity of NP was evaluated through micronuclei assay and single cell gel electrophoresis (Comet assay) in peripheral erythrocytes of Oreochromis niloticus exposed in vivo. The study on reproductive development was also carried out in male and female gonads of O. niloticus. Lethal concentration (LC 50%) of 0.032 ml l⁻¹ was previously determined. We ran assays with O. niloticus exposed to concentrations of 1.0, 10.0, and 16.0 μl l⁻¹ of NP diluted in water. Our results showed that NP was not genotoxic. However, 3-day exposure to NP in concentrations of 1.0, 10.0, and 16.0 μl l⁻¹ of water increased the frequency of reproductive stages in males and females. The histology of the reproductive tract of the treated fish was significantly altered in females treated with 16.0 μl l⁻¹ of water when compared to controls. Analogous estrogenic effects were observed, such as accelerated maturation of oocytes and spermatogenesis. These results showed that the O. niloticus reproductive system is sensitive to NP estrogenicity.