The effects of dietary nickel exposure on growth and reproduction of Daphnia magna

Although there is growing evidence that dietborne metals can be toxic to various aquatic species, there is still insufficient knowledge to integrate this information in environmental risk assessment procedures. In this study, we investigated the effects of a 21-day exposure of Daphnia magna to a control diet (i.e. the green alga Pseudokirchneriella subcapitata containing <4.0 μg Ni/g dry wt) and five diets with elevated Ni concentrations (i.e. the same alga contaminated with Ni burdens between 33.7 and 837 μg Ni/g dry wt). A significant accumulation of dietborne Ni in D. magna, i.e. between 49.6 and 72.5 μg Ni/g dry wt, was observed when they were fed with diets containing between 85.6 and 837 μg Ni/g dry wt. This was paralleled by a significant reduction of reproduction (by 33.1%), measured as the total number of juvenile offspring per female and growth (by 9.1%), measured as the carapax length of 21-day-old females. Life-history analysis showed that the time to first brood of Ni exposed organisms was between 7.8 and 8.2 days, and occurred 0.7–1.1 days earlier than for the control organisms (time to first brood = 8.9 days). The number of offspring in the first brood was significantly reduced (by 21–33% compared to the control) in all dietary treatments. Longer exposure (≥8.9 days, i.e. from the second brood onwards) led to a reduction of brood size only when given diets containing 85.6 and 837 μg Ni/g dry wt. The results suggest that a variety of mechanisms may be involved in the effects of dietary Ni exposure, including altered resource allocation or targeted reproductive inhibition. While Ni exposure clearly altered the quality of the diet (measured as essential ω3 polyunsaturated fatty acid content and C:P ratio), we found no conclusive evidence that these diet quality shifts could have affected growth or total reproductive output. More research is required to fully understand the mechanisms of Ni toxicity associated with the dietary exposure route.     

Separation and determination of coumarins in Fructus cnidii extracts by pressurized capillary electrochromatography using a packed column with a monolithic outlet frit

The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15 min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at −6 kV applied voltage. The calibration curves were linear in the range of 10.0–100.0 μg/mL for bergapten, 20.0–200.0 μg/mL for imperatorin, 5.0–400.0 μg/mL for osthole, 10.0–100.0 μg/mL for 2′-acetylangelicin, 10.0–200.0 μg/mL for oroselone, and 10.0–200.0 μg/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Magnolia liliflora Desr.

This study was carried out to assess the antioxidant and antidermatophytic activities of the essential oil and extracts of Magnolia liliflora Desr. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC₅₀ values = 10.11 and 16.17 μg/ml, respectively) as compared to butylatedhydreoxyanisole (BHA), (IC₅₀ value = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (96.13 mg/g of dry wt) as compared to the other extracts. Further, the oil (1000 μg/disc) and extracts (1500 μg/disc) revealed 42.36–63.12% and 19.07–54.14% antidermatophytic effect, respectively along with their respective MIC values ranging from 62.5 to 500 and 250 to 2000 μg/ml against the members of Trichophyton and Microsporum spp. Also the oil had strong detrimental effect on spore germination of tested fungal pathogens as well as concentration and time dependent kinetic inhibition of Microsporum canis KCTC 6348. The results of this study justify a potential role of M. liliflora to serve as a natural antioxidant and antidermatophytic agent.     

In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 μg/ml) was increased in a dose dependent manner, which was found in the range of 36.93–68.76% as compared to ascorbic acid 64.26–82.58%. The IC₅₀ values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 93.46 and 26.75 μg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of S. rebaudiana was achieved using Folin–Ciocalteau reagent containing 61.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC₅₀ values of 93.46, 132.05 and 81.08 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 μg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.     

Amnesic shellfish poisoning toxins in bivalve molluscs in Ireland

In December 1999, domoic acid (DA) a potent neurotoxin, responsible for the syndrome Amnesic Shellfish Poisoning (ASP) was detected for the first time in shellfish harvested in Ireland. Two liquid chromatography (LC) methods were applied to quantify DA in shellfish after sample clean-up using solid-phase extraction (SPE) with strong anion exchange (SAX) cartridges. Toxin detection was achieved using photodiode array ultraviolet (LC-UV) and multiple tandem mass spectrometry (LC-MSⁿ). DA was identified in four species of bivalve shellfish collected along the west and south coastal regions of the Republic of Ireland.

The amount of DA that was present in three species was within EU guideline limits for sale of shellfish (20 μg DA/g); mussels (Mytilus edulis), <1.0 μg DA/g; oysters (Crassostrea edulis), <5.0 μg DA/g and razor clams (Ensis siliqua), <0.3 μg DA/g. However, king scallops (Pecten maximus) posed a significant human health hazard with levels up to 240 μg DA/g total tissues. Most scallop samples (55%) contained DA at levels greater than the regulatory limit. The DA levels in the digestive glands of some samples of scallops were among the highest that have ever been recorded (2820 μg DA/g).     

Development of ultra fast liquid chromatographic method for simultaneous determination of nitrendipine and carvone in skin diffusate samples

A simple and sensitive reverse phase ultra fast liquid chromatographic (UFLC) method for simultaneous determination of nitrendipine and carvone in skin diffusate samples and microemulsions was developed and validated. The separation was achieved using a gradient mobile phase, on an Onyx column. The eluents were monitored by photodiode array detection. The linearity ranges of proposed method were 0.125–50 μg mL⁻¹ and 0.125–30 μg mL⁻¹ for nitrendipine and carvone respectively. The intra-day and inter-day coefficient of variation and percent error values of the assay method were less than 10%. The method was found to be precise, accurate, and specific during the study. The method was successfully applied for simultaneous estimation of nitrendipine and carvone in ex vivo skin diffusate samples and microemulsions.     

LC-MS/MS method for determination of hederacolchiside E, a neuroactive saponin from Pulsatilla koreana extract in rat plasma for pharmacokinetic study

A simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was applied to pharmacokinetic study of a neuroactive oleanolic-glycoside saponin, hederacolchiside E from SK-PC-B70M, a standardized extract of Pulsatilla koreana in rat. Rat plasma samples were pretreated by protein precipitation with acetonitrile, eluted from C₁₈ column, and analyzed using electrospray ionization (ESI)-MS/MS in negative ion mode. Digoxin was used as an internal standard. The standard curves were linear (r > 0.997) over the concentration ranges of 2–500 ng/mL. The intra- and inter-day precisions were measured to be below 9% and accuracy between 90 and 111% for all quality control samples at 2, 20, 100, and 500 ng/mL (n = 5). The lower limits of quantification (LLOQ) for hederacolchiside E was 2 ng/mL and the limit of detection (LOD) 0.5 ng/mL using 20 μL of plasma sample. Subsequently, hederacolchiside E was determined in rat plasma samples after oral administration of SK-PC-B70M. The mean maximum plasma concentrations of hederacolchiside E were 0.07, 0.13, and 0.36 μg/mL and the mean areas under the plasma concentration versus time curve 0.56, 1.27, and 6.46 μg h/mL at doses of 100, 200, and 400 mg/kg, respectively, which indicated non-linear pharmacokinetic pattern. In conclusion, this method was successfully applied to the pharmacokinetic study of hederacolchiside E after an oral administration of SK-PC-B70M to rats.     

In vitro analyses of the combination of high-dose doxycycline and antifungal agents against Candida albicans biofilms

The potential of antifungal agents used as antimicrobial lock therapy (ALT) for the conservative management of catheter-related candidemia has not been fully defined. We sought to determine the antifungal effect of high-dose doxycycline (DOX), alone or in combination with standard concentrations of amphotericin B (AMB), caspofungin (CAS) or fluconazole (FLC), against biofilms formed by Candida albicans in vitro. DOX alone (at 2048 μg/mL and 1024 μg/mL) demonstrated up to an 85% reduction of the metabolic activity of the C. albicans biofilm. Regardless of the concentration tested, FLC alone showed minimal activity (mean 22.9% reduction) against the C. albicans biofilm. When DOX 2048 μg/mL was used in combination with FLC, antifungal activity also increased up to 85%, suggesting an additive effect. DOX 128 μg/mL in combination with FLC demonstrated synergy (mean 58.3% reduction). The combination of DOX 2048 μg/mL or 512 μg/mL and AMB was superior to AMB alone at low concentrations (0.25–0.03125 μg/mL). However, DOX 128 μg/mL was antagonistic in combination with low concentrations of AMB. Maximal efficacy against the biofilm was observed with CAS at 8–0.25 μg/mL compared with FLC and AMB alone. A paradoxical effect (PE) occurred with CAS at 16 μg/mL, which showed a marked reduction in antifungal activity compared with lower concentrations of CAS. CAS at 16 μg/mL in combination with either DOX 2048 μg/mL or 512 μg/mL resulted in attenuation of the PE. These findings suggest that a high-dose DOX-based ALT strategy in combination with traditional antifungal agents may be useful for the treatment of C. albicans biofilms.     

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 3² factorial design in electrolytes consisting of sodium tetraborate buffer (STB) in the concentration range of 10–50 mmol L⁻¹ and methanol in the volume percentage of 5–20%. Derringer's desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L⁻¹ tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid–liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 μg mL⁻¹, R² > 0.999; LOD and LOQ from 1.32 to 3.80 μg mL⁻¹ and from 4.01 to 11.5 μg mL⁻¹, respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8–11% CV for peak area; recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50–47.3 μg g⁻¹ were found, with smaller amounts occurring as free phenolic acids.     

A sensitive LC–MS/MS method to quantify loureirin B in rat plasma with application to preclinical pharmacokinetic studies

A novel method for the quantification of loureirin B in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) was developed. Loureirin B and internal standard (buspirone) were extracted by liquid–liquid extraction and separated on a Agilent XDB C₁₈ column (50 mm × 4.6 mm, 5 μm). As mobile phase a binary mixture of methanol (containing 0.1% formic acid)–water (containing 0.1% formic acid) was delivered by a Shimadzu LC-20AD pump in gradient mode at a flow rate of 0.4 ml/min in a run time of 5.0 min. The detector was a Q-trap™ mass spectrometer with an electrospray ionization (ESI) interface operating in the multiple reaction monitoring (MRM) mode. The calibration curve of loureirin B in plasma showed good linearity over the concentration range of 0.08–100 ng/ml. The limit of detection and limit of quantification were 0.03 ng/ml and 0.08 ng/ml, respectively. Intra- and inter-day precisions (as relative standard deviation) in all samples were both within 15%. The validated method was successfully applied to a preliminary pharmacokinetic study of loureirin B in rats. After oral administration of 16 g/kg longxuejie to rats, the main pharmacokinetic parameters t_max, C_max, t_1/2, K_e and AUC_0–T were 0.8 h, 7.99 μg/l, 1.94 l h, 0.365/h, and 22.21 μg h/l, respectively.     

Development and validation of a capillary electrophoresis method for the simultaneous determination of impurities of escitalopram including the R-enantiomer

A stereospecific capillary electrophoresis assay for the simultaneous determination of related substances and the enantiomeric purity of escitalopram was developed by a central composite face-centered factorial design and subsequently validated. Separations were carried out in a 50 μm, 47/40 cm fused-silica capillary. The optimized conditions included 20 mM phosphate buffer, pH 2.5, containing 0.5 mg/ml β-cyclodextrin and 22 mg/ml sulfated β-cyclodextrin as background electrolyte, an applied voltage of −20 kV and a temperature of 28 °C. Salicylic acid was used as internal standard. The assay was validated for the (R)-enantiomer of citalopram and the enantiomers of the impurity citadiol in the range of 2.5–150 μg/ml and 2.5–50 μg/ml, respectively. The limit of detection was 0.02% for all compounds, the limit of quantitation 0.05%, relative to a concentration of escitalopram of 5 mg/ml. Intraday precision of migration time and peak area ratio were in the range of 0.17–0.44% and 1.64% and 6.25%, respectively. Relative standard deviations of interday precision ranged between 0.84% and 1.85% in the case of migration times and between 5.20% and 9.28% for peak area ratio. The assay was applied to the determination of the purity of escitalopram in bulk drug and tablets. (R)-Citalopram and (S)-citadiol were detected as impurities.     

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27° SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer.In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels.Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160 μg/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.     

Chemical composition and cytotoxic, mutagenic and genotoxic activities of the essential oil from Piper gaudichaudianum Kunth leaves

We have investigated the chemical composition of Piper gaudichaudianum essential oil, as well as its cytotoxic, mutagenic and genotoxic effects in V79 cells. The chemical analyses showed that the major compounds are (E)-nerolidol (22.4%), α-humulene (16.5%), (E)-caryophyllene (8.9%) and bicyclogermacrene (7.4%). Dose-dependent cytotoxic effects were observed in V79 cells treated with essential oil by using clonal survival, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction assay (MTT) and trypan blue exclusion assay (TB), and a significant decrease in survival was observed at concentrations of 0.5 μg/mL and higher. The P. gaudichaudianum essential oil treatment caused DNA strand breaks in V79 cells at concentrations up to 2 μg/mL, as detected by the alkaline comet assay, but did not induce double-strand breaks, as verified by neutral comet assay. It induced a significant increase in the frequency of micronucleated cells at 4, 6 and 10 μg/mL. Moreover, P. gaudichaudianum essential oil significantly increased lipid peroxidation at doses of 0.5 μg/mL and higher, suggesting that the observed oxidant potential can be responsible, at least in part, for its cytotoxic and genotoxic effects.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.     

Mechanisms of p,p′-DDE-induced apoptosis in human peripheral blood mononuclear cells

p,p′-Dichlorodiphenyldichloroethylene (DDE), the most stable metabolite of organochlorine insecticide p,p′-dichlorodiphenyltrichloroethane (DDT), has been detected in human populations living in malaria-endemic areas of México where this insecticide was used. DDE induces apoptosis in peripheral blood mononuclear cells (PMBC); however, the molecular mechanism of cell death induced by this compound is poorly understood. In the present study, PBMC isolated from healthy individuals (not exposed to DDE) were incubated in the presence of increasing concentrations of p,p′-DDE (0–80 μg/ml) over time. When PBMC were treated with low p,p′-DDE concentration (10 μg/ml) an antioxidant response and biomarkers of inflammation were induced, indicating a pro-inflammatory state. Moreover, when PBMC were treated with high p,p′-DDE concentration (80 μg/ml) several apoptotic biochemical events were triggered, such as activation of caspase-8, Bid, caspase-9 and caspase-3, as well as degradation of PARP and ubiquitination. The results described in this study show a possible inflammatory condition and the involvement of both extrinsic and intrinsic pathways in the induction of apoptosis in DDE-treated PBMC.     

Studies on the antioxidant activity of essential oil and different solvent extracts of Vitex agnus castus L. fruits from Turkey

This study is designed to examine the chemical composition and antioxidant activity of the essential oil and different solvent extracts of Vitex agnus castus. GC and GC–MS analysis was resulted in the detection of 27 components, representing 94.5% of the oil. Major components of the oil were 1,8-cineole (24.98%), sabinene (13.45%), α-pinene (10.60%), α-terpinyl acetate (6.66%), and (Z)-β-farnesene (5.40%). Antioxidant activities of the samples were determined by three different test systems, DPPH, β-carotene/linoleic acid and reducing power assays. In all systems, water extract exhibited excellent activity potential than those of other extracts (hexane, dichloromethane, ethyl acetate and methanol) and the oil. As expected, amount of total phenolics was very high in this extract (112.46 ± 1.22 μg GAEs/mg extract). Dichloromethane extract has been found to be rich in flavonoids. A positive correlation was observed between the antioxidant activity potential and total phenolic and flavonoid levels of the extracts.     

Steady-state pharmacokinetics and pharmacodynamics of piperacillin/tazobactam administered by prolonged infusion in hospitalised patients

The objective of this study was to evaluate the steady-state pharmacokinetics and pharmacodynamics of piperacillin/tazobactam, administered by prolonged infusion, in hospitalised patients requiring antimicrobial therapy. Thirteen patients received 4.5 g every 8 h (q8h), infused over 4 h, and pharmacokinetic parameters were determined by non-compartmental methods. Monte Carlo simulations (10 000 patients) were performed to calculate the cumulative fraction of response (CFR) for seven Gram-negative pathogens using minimum inhibitory concentration (MIC) data from the Meropenem Yearly Susceptibility Test Information Collection (2004–2007, USA) as well as the probability of target attainment (PTA) at MICs ranging from 1 μg/mL to 64 μg/mL. The pharmacodynamic target was free piperacillin concentration remaining above the MIC for 50% of the dosing interval. Mean ± standard deviation maximum and minimum serum concentrations, half-life, volume of distribution at steady-state and systemic clearance of piperacillin were 108.2 ± 31.7 μg/mL, 27.6 ± 26.3 μg/mL, 2.1 ± 1.2 h, 22.1 ± 4.0 L and 8.6 ± 3.0 L/h, respectively. The CFR was >90% for Escherichia coli, Serratia marcescens and Citrobacter spp., 88.6% for Enterobacter spp., 87% for Klebsiella pneumoniae, 85.5% for Pseudomonas aeruginosa and 52.8% for Acinetobacter spp. The PTA was 100%, 81.1% and 12.3% at MICs of ≤16 μg/mL, 32 μg/mL and 64 μg/mL, respectively. Piperacillin/tazobactam 4.5 g q8h infused over 4 h provides excellent target attainment for bacterial pathogens with MICs ≤ 16 μg/mL. However, the CFR was <90% for four of the seven Gram-negative pathogens evaluated.     

Oxidative stress, protein carbonylation and heat shock proteins in the black tiger shrimp, Penaeus monodon, following exposure to endosulfan and deltamethrin

The impact of commonly used pesticides, endosulfan and deltamethrin, on the molecular stress level in black tiger shrimp Penaeus monodon, was assessed using classical oxidative stress biomarkers, protein carbonylation profiles, and levels of heat shock proteins. Results showed that 4 days exposure to 0.1 μg L⁻¹ deltamethrin significantly (p < 0.05) increased lipid peroxidation (LPO) level in gills (64.3 ± 3.2 compared to 34.2 ± 5.3 nmol MDA equiv. g⁻¹ tissue at day 0). However, no pesticide treatment had significant effect on the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Carbonylated protein profiles were determined on gills following 2,4-dinitrophenylhydrazine derivatization and 2D-PAGE along with Western blotting. Immunoblotting with dinitrophenol-specific antibody revealed 17 protein spots carbonylated in response to 4 days exposure to 0.1 μg L⁻¹ deltamethrin while 24 protein spots specifically oxidized at day 0 were no longer detected after deltamethrin treatment. On the other hand, endosulfan exposure at 0.1 and 1 μg L⁻¹ induced up to 2.1-fold increase of HSP90 level in muscle. This approach is providing new insights into the molecular impacts of deltamethrin and endosulfan on an economically important crustacean. While deltamethrin has shown a pro-oxidant effect in gills, endosulfan exposure rather induced proteotoxic effects in muscles. This argues that LPO level, protein carbonylation specificities, and HSP90 levels may be potential discriminating biomarkers to assess the chemical stress level in farm shrimp.     

Developmental anomalies induced by a non-selective COX inhibitor (ibuprofen) in zebrafish (Danio rerio)

Effects of ibuprofen (a non-selective COX inhibitor) on the embryonic development, hatching success, larval growth, behavioral pattern and survival competence were studied in Danio rerio. Embryos at 2/4 celled stage were exposed to graded doses (0, 1, 5, 10, 50 and 100 μg/L distilled water) of ibuprofen in triplicate sets (n = 30). The experiment was repeated thrice. The results indicate that developing embryos tolerated lower (1 and 5 μg/L) doses of the drug readily but, exposure to higher doses (>10 μg/L) caused retarded development, decreased hatching rate and growth, cardiac anomalies, spinal curvature, pectoral fin malformation and behavioral alterations resulting in greater mortality of experimental embryos. This study suggests that, ibuprofen which is marketed as over-the-counter (OTC) drug is embryotoxic at least at higher (>10 μg/L) dose level to zebrafish embryos.     

Cypermethrin-induced histopathological and biochemical changes in Nile tilapia (Oreochromis niloticus), and the protective and recuperative effect of ascorbic acid

The objective of this study was to investigate the effects of ascorbic acid on the toxicity of cypermethrin's on histopathological lesions in tissues and protein, glycogen levels in Oreochromis niloticus. Nile tilapia was exposed to 0.22 and 0.44 μg/l cypermethrin + control diet, 0.22 and 0.44 μg/l cypermethrin + ascorbic acid supplemented diet for 20 days. The fish were allowed recuperation period of 15 days in pesticide-free water and fed with ascorbic acid suplementation diet. In light microscopic investigation, histopathological lesions were observed in the gill, liver and kidney. The severity of lesions accreted depending on increased pesticide concentration and control diet. Some of the lesions were reversible or at least were less pronounced after recuperation period. Protein levels decreased in some groups after treatment period according to control groups (p < 0.05). The highest depletions in liver, muscle and gill protein levels were found in 0.44 μg/l cypermethrin + ascorbic acid supplemented diet group (62.23%), in 0.22 μg/l cypermethrin + control diet group (53.12%) and in 0.44 μg/l cypermethrin + control diet group (61.87%) after 10 days, respectively. These levels increased at the end of the recuperation period. The highest depletion in liver glycogen levels was found in 0.22 μg/l cypermethrin + control diet group (50.50%) after 10 days (p < 0.05). At the end of recuperation period, there was no difference between the groups (except 0.22 μg/l cypermethrin + ascorbic acid supplemented diet group) and controls. The decrease of muscle glycogen, except 0.22 μg/l cypermethrin + ascorbic acid supplemented diet group, was recorded at the end of 10 and 20 days. In the recuperation period, an increase was observed at all groups. These results revealed that the histopathology, protein and glycogen can work as good indicators of stress of a toxicant on fish. Ascorbic acid serves fish as an antitoxic agent against pesticide toxicity.