Impairment of trophic interactions between zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) and midge larvae (<Emphasis Type="Italic">Chironomus riparius</Emphasis>) by chlorpyrifos

The effects of chemicals on biotic interactions, such as competition and predation, have rarely been investigated in aquatic ecotoxicology. This study presents a new approach for the investigation of predator–prey interactions between zebrafish (Danio rerio) and midge larvae (Chironomus riparius) impaired by chlorpyrifos (CHP), a neurotoxic insecticide. With a simple experimental design including four different treatments: (1) control, (2) predator exposed, (3) prey exposed and (4) both, predator and prey, exposed, we were able to detect by visual observation an increase in the feeding rate of zebrafish preying on exposed chironomids after acute (2 h) exposure to 6 μg/l CHP. Previously, a decrease in the burrowing behaviour of exposed chironomid larvae was observed. However, when pre-exposing simultaneously both predators and prey, no significant differences in the feeding rate of zebrafish were observed. This suggests an impairment in prey recognition of the exposed zebrafish. At a lower CHP concentration (1 μg/l), no differences in feeding rate of zebrafish were observed. We therefore propose the use of trophic interactions as parameters in higher tier studies for chemical testing and evaluation of ecotoxicological risk assessment.     

Acute toxic and genotoxic effects of formalin in <Emphasis Type="Italic">Danio rerio</Emphasis> (zebrafish)

Formalin is a readily soluble chemical used as a sanitizing agent in the home and hospital. Formaldehyde solutions are routinely used in aquaculture for the prophylaxis and treatment of parasites and fungi, but the adverse effects of their application need to be further investigated. Danio rerio or zebrafish has characteristics favorable to its handling and breeding, and it is highly sensitive to various chemicals, being an ideal experimental model for this type of investigation. Thus, the objective of this study was to verify the toxic and genotoxic effects of formalin and to determine the lethal concentrations of this chemical to support its safe use in disinfection processes. Acute and chronic tests were performed using methods in accordance with international protocols. The genotoxic effect of formalin was evaluated with the micronucleus test using blood samples, which were collected at 96 and 192?h of exposure. The LC_50–96h of formalin in D. rerio was 45.73?mg?L^?1, demonstrating its high resistance compared to other species. Regarding the genotoxic effect, the sublethal concentrations of formalin showed a positive correlation with micronuclei according to the increase in its concentration independent of the time of exposure. The incidence of micronuclei increased with concentration, and the addition of 1?mg?L^?1 formalin corresponded to an increase of 2.9% in the average number of micronuclei. In other words, formalin at even sublethal concentrations caused genotoxic effects in peripheral blood erythrocytes of D. rerio. Therefore, we recommend further studies and other tests involving this chemical for its use at environmentally safe concentrations.     

Stress test of a biological early warning system with zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

The aim of this work was to develop a novel methodology to stress test the diagnostic capability of a video tracking system with zebrafish (Danio rerio), against two pre-established disturbances. Eight different treatments were tested varying the presence or absence of a toxicant (NaOCl) and two disturbances: the passing of a shadow (mimicking a predator) and entrapment of the fish. The concentration tested corresponded to a sublethal (1?% 24?h-LC₅₀) and short term exposure (2?h). A total of 56 organisms were tested resulting in 112 diagnoses (before and after the contamination). A statistical model of diagnosis was developed using Self-organizing Map (SOM) and Correspondence Analysis (CA). Sensitivity, specificity, accuracy, false positive and false negative values were calculated to evaluate the diagnostic performance. The disturbances did not negatively affect the capability of the model. In the presence of at least one of these variables, the diagnostic performance was similar or even superior to the baseline results without disturbances. Furthermore, the system produced a large number of correct diagnoses, at an ecologically relevant concentration of exposure, in a non-invasive way.     

Methyl-triclosan and triclosan impact embryonic development of <Emphasis Type="Italic">Danio rerio</Emphasis> and <Emphasis Type="Italic">Paracentrotus lividus</Emphasis>

The presence of emerging pollutants in the environment is of major concern not only because of the potential negative impact in human health, but also due to the potential toxicity to non-target organisms. Within the personal and care products (PCPs), the disinfectant Triclosan (TCS) is one of the most concerning compounds. Once in the wastewater treatment plants (WWTPs), a small part of TCS can be biotransformed into a more persistent by-product: methyl-triclosan (M-TCS). Although several studies have focused on the occurrence of this compound in the water systems, the information on its toxicity to aquatic organisms is very limited. Here, we used embryo bioassays with two aquatic model animals to improve risk assessment of M-TCS; zebrafish (Danio rerio) embryo bioassays run up to 144?h post fertilization (hpf) and sea urchin (Paracentrotus lividus) up to 48?hpf, following established protocols. M-TCS and TCS exhibited similar toxicity to zebrafish with a NOEC of 160?µg/L. In contrast, M-TCS induced a delay in the development of the sea urchin larvae at all tested concentrations (1–1000?µg/L), whereas NOEC of TCS for P. lividus embryos was 40?µg/L. Overall, given the reported effects of M-TCS in the close range of environmentally relevant concentrations, and considering the low degradation rate and tendency to bioaccumulation (logKow: 5.2), further studies are warrant to better characterize the risk of this TCS metabolite to aquatic organisms.     

Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) in a partial life-cycle test

Toxicological effects of the widely used flame retardant, tetrabromobisphenol A (TBBPA) were assessed in a partial life-cycle test with zebrafish (Danio rerio). Exposure of adult fish during 30 days to water-borne TBBPA in nominal concentrations ranging from 0 (control) to 1.5 μM was followed by exposure of the offspring in early life stages up to 47 days posthatching (dph) to the same concentrations. Adults exposed to 3 and 6 μM showed severe disorientation and lethargy shortly after beginning of exposure and were euthanized. Because semistatic exposure resulted in fluctuating water concentrations, pooled fish samples were chemically analyzed for internal dose assessment. Egg production was decreased in fish exposed to TBBPA concentrations of 0.047 μM and higher, and a critical effect level of 7.2 μg/g lipid with a lower 5% confidence limit of 3.9 μg/g lipid for 50% decreased egg production was calculated. Histology of adult ovaries indicated a relative increase of premature oocytes in two surviving females exposed to 1.5 μM. Hatching of TBBPA-exposed larvae was decreased except in animals exposed to 0.375 μM. In the highest exposure concentration, early posthatching mortality was high (81%) in larvae and the surviving juveniles showed a significant predominance of the female phenotype. Exposure of eggs from control parents up to 6 μM TBBPA resulted in increasing malformation and pericardial fluid accumulation from 1.5 μM; at higher concentrations, all embryos failed to hatch. The presented results indicate decreased reproductive success in zebrafish at environmentally relevant TBBPA concentrations.     

Joint toxicity of cadmium and SDBS on <Emphasis Type="Italic">Daphnia magna</Emphasis> and <Emphasis Type="Italic">Danio rerio</Emphasis>

Information on joint toxicity is limited. To clarify the joint toxicity and the interactions among toxicants on different aquatic organisms, we investigated the acute toxicity of cadmium and sodium dodecyl benzene sulfonate, two chemicals with high concerns in Chinese waters, on the immobilization of Daphnia magna (D. magna) and the swimming behavior of Danio rerio (D. rerio). Our results illustrated that cadmium and sodium dodecyl benzene sulfonate expressed a synergistic effect on the immobilization of D. magna; and an antagonistic effect on the swimming speed D. rerio, but a synergistic effect on its vertical position in the water column. Based on the observed data, we found the independent action model was more appropriate than the concentration addition model in the prediction of their joint toxicity. Our results gave an example of the joint toxicity investigation, and aided to comprehensive the toxicity action mode of chemical mixtures.     

Effect of proton beam on blood vessel formation in early developing zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos

Proton beam therapy can kill tumor cells while saving normal cells because of its specific energy delivery properties and so is used to various tumor patients. However, the effect of proton beam on angiogenesis in the development of blood vessels has not been determined. Here we used the zebrafish model to determine in vivo whether proton beam inhibits angiogenesis. Flk-1-GFP transgenic embryos irradiated with protons (35 MeV, spread out Bragg peak, SOBP) demonstrated a marked inhibition of embryonic growth and an altered fluorescent blood vessel development in the trunk region. When cells were stained with acridine orange to evaluate DNA damage, the number of green fluorescent cell death spots was increased in trunk regions of irradiated embryos compared to non-irradiated control embryos. Proton beam also significantly increased the cell death rate in human umbilical vein endothelial cells (HUVEC), but pretreatment with N-acetyl cystein (NAC), an antioxidant, reduced the proton-induced cell death rate (p<0.01). Moreover, pretreatment with NAC abrogated the inhibition of trunk vessel development and prevented the trunk malformation caused by proton irradiation. In conclusion, proton irradiation significantly inhibited in vivo vascular development possibly due to increased vascular cell death via reactive oxygen species formation.     

<Emphasis Type="Italic">NAT2</Emphasis>,<Emphasis Type="Italic"> CYP2C9</Emphasis>,<Emphasis Type="Italic"> CYP2C19</Emphasis>, and<Emphasis Type="Italic"> CYP2E1</Emphasis> genetic polymorphisms in anti-TB drug-induced maculopapular eruption

Purpose  

It has been suggested that drug-metabolizing enzymes might play important roles in the development of anti-tuberculosis drug (ATD)-induced maculopapular eruption (MPE), as in ATD-induced hepatitis. We investigated the associations between the genetic polymorphisms of drug-metabolizing enzymes and ATD-induced MPE.     

The toxic potential of aldrin and heptachlor on <Emphasis Type="Italic">Danio rerio</Emphasis> juveniles (Cypriniformes,Cyprinidae)

With the objective of evaluating the effects of organochlorine pesticides (aldrin and heptachlor) on the survival, growth and gill morphology of juvenile zebrafish (Danio rerio), four partial chronic toxicity bioassays were conducted (seven days’ duration) with both compounds in a semi-static system with renewal every 24 h. The results did not show any effects on the fish’s survival, but did on their growth and gill morphology. Aldrin was more toxic than heptachlor, since the chronic value (CV) for growth was nearly 8.7 times less, a result confirmed by analyzing the histology of the gills, in which the changes detected were considered more severe for the former substance. In general, they were found branchial lesions of first stage, in other words, cell proliferation between the secondary lamellae, hyperplasia, lifting of respiratory epithelial cells; fusion of several secondary lamellae and dilation of blood vessels. Aneurysms (alterations of second stage) they were verified in the exposed organisms to the aldrin, which also presented about 10% of reduction in the standard length and 30% of reduction in the total weight in relation to the exposed organisms to the control. For heptachlor these values were of 8% and 25%, respectively. The intensity of the gill lesions and growth of the fish did not depend on the pesticide concentration, suggesting different modes of action of the products.     

Genetic and chemical comparison of Boi (<Emphasis Type="Italic">Sinomeni Caulis</Emphasis><Emphasis Type="Italic">et Rhizoma</Emphasis>) and Seifuto (<Emphasis Type="Italic">Caulis Sinomenii</Emphasis>)

Boi and its original plant Sinomenium acutum from Japan were compared with Seifuto and its botanical origins from China in terms of their internal transcribed spacer (ITS) sequences and major chemical components. Boi, Seifuto, and their botanical origins overall showed seven variable sites in the ITS sequence and six genotypes. Japanese S. acutum and Boi had one nucleotide variation at position 593 to show two genotypes (J1 and J2) and their heterozygote (J3). Seifuto samples and their botanical origins, S. acutum and S. acutum var. cinereum from China, showed three genotypes (C1, C2, and C3), which did not agree with the botanical classification, indicating that they cannot be distinguished according to their ITS sequences. All Seifuto samples from Henan market showed the same ITS genotype (C1). The Japanese and Chinese genotypes differed in the nucleotide position 424, which can be used to distinguish the country of origin of these materials. In the HPLC analysis of six major components, sinomenine (1), magnoflorine (2), menisperine (3), 6-O-methyllaudanosoline glucoside (4), liriodendrin (5), and menisdaurin (6), all were detected in Boi, whereas five (all except for menisdaurin) were detected in Seifuto. The main component in the rhizome of Seifuto was sinomenine, whereas magnoflorine was the main component in the rhizome and the climbing stem of Boi. The content of sinomenine in Seifuto was almost twice that in Boi. Although the individual content of alkaloids 1–4 differed between Boi and Seifuto, the total contents of these alkaloids were comparable between them both in the climbing stem and rhizome.     

Mild hypoglycaemic attacks induced by sulphonylureas related to <Emphasis Type="Italic">CYP2C9</Emphasis>, <Emphasis Type="Italic">CYP2C19</Emphasis> and <Emphasis Type="Italic">CYP2C8</Emphasis> polymorphisms in routine clinical setting

Aim  

To evaluate the impact of polymorphisms in the cytochrome P450 (CYP) 2C9, 2C19 and 2C8 genes on the risk of mild hypoglycaemic attacks in patients treated with sulphonylureas.     

<Emphasis Type="Italic">CYP2D6*4</Emphasis>, <Emphasis Type="Italic">CYP3A5*3</Emphasis> and <Emphasis Type="Italic">ABCB1 3435T</Emphasis> polymorphisms and drug-related falls in elderly people

Objective The objective of this study is to investigate the association between CYP2D6*4, CYP3A5*3 and ABCB1 3435T polymorphisms and drug-related falls. Method Multivariate logistic regression was performed in an existing database in order to study the association between falls history and CYP2D6*4, CYP3A5*3, ABCB1 3435T polymorphisms in patients using fall-risk-increasing CYP2D6, CYP3A5 and P-glycoprotein (gene product of ABCB1) substrates. Results No statistically significant increased fall risk was found in ‘poor metabolizers’ compared to ‘extensive’ and ‘intermediate metabolizers’ using fall-risk-increasing CYP2D6 substrates (Odds ratio (OR) = 2.2; 95% confidence interval (CI) 0.2–25.0), CYP3A5 substrates (OR = 0.9; 95% CI 0.2–3.3) and P-glycoprotein substrates (OR = 2.1; 95% CI 0.2–17.2). Conclusion The hypothesis that ‘poor metabolizers’ have an increased fall risk was not confirmed. A larger study population is needed to confirm the potential association that was seen between CYP2D6*4 and ABCB1 3435T polymorphisms and drug-related falls.     

<Emphasis Type="Italic">ent</Emphasis>-Abietane diterpenoids from <Emphasis Type="Italic">Isodon xerophilus</Emphasis>

Three new ent-abietanoids, named xerophilusins XIV–XVI, and four known analogues, as well as four known chemical constituents were isolated from the leaves of Isodon xerophilus. Their structures were elucidated by extensive spectroscopic studies, and comparison with literature data. In addition, the cytotoxic activity of the ent-abietanoids against chronic myelogenous leukemia (K562), stomach adenocarcinoma (MKN45), and hepatocellular carcinoma (HepG2) human cell lines was investigated and no activities were observed.     

Identification of medicinal <Emphasis Type="Italic">Dendrobium</Emphasis> species by phylogenetic analyses using <Emphasis Type="Italic">matK</Emphasis> and <Emphasis Type="Italic">rbcL</Emphasis> sequences

Species identification of five Dendrobium plants was conducted using phylogenetic analysis and the validity of the method was verified. Some Dendrobium plants (Orchidaceae) have been used as herbal medicines but the difficulty in identifying their botanical origin by traditional methods prevented their full modern utilization. Based on the emerging field of molecular systematics as a powerful classification tool, a phylogenetic analysis was conducted using sequences of two plastid genes, the maturase-coding gene (matK) and the large subunit of ribulose 1,5-bisphosphate carboxylase-coding gene (rbcL), as DNA barcodes for species identification of Dendrobium plants. We investigated five medicinal Dendrobium species, Dendrobium fimbriatum, D. moniliforme, D. nobile, D. pulchellum, and D. tosaense. The phylogenetic trees constructed from matK data successfully distinguished each species from each other. On the other hand, rbcL, as a single-locus barcode, offered less species discriminating power than matK, possibly due to its being present with little variation. When results using matK sequences of D. officinale that was deposited in the DNA database were combined, D. officinale and D. tosaense showed a close genetic relationship, which brought us closer to resolving the question of their taxonomic identity. Identification of the plant source as well as the uniformity of the chemical components is critical for the quality control of herbal medicines and it is important that the processed materials be validated. The methods presented here could be applied to the analysis of processed Dendrobium plants and be a promising tool for the identification of botanical origins of crude drugs.     

A new <Emphasis Type="Italic">Erythrina</Emphasis> alkaloid from <Emphasis Type="Italic">Erythrina herbacea</Emphasis>

A new Erythrina alkaloid, 10-hydroxy-11-oxoerysotrine (1), has been isolated from the flowers of Erythrina herbacea together with five known compounds: erytharbine (2), 10,11-dioxoerysotrine (3), erythrartine (4), erysotramidine (5) and erysotrine-N-oxide (6). The structure of the new compound was elucidated on the basis of its spectral data, including 2-D NMR and mass (MS) spectra. The new compound is a rare C-10 oxygenated Erythrina alkaloid. The antioxidant activities of the isolated compounds 1–6 were evaluated by scavenging with peroxynitrite.     

<Emphasis Type="Italic">In Vitro</Emphasis> hepatoprotective compounds from <Emphasis Type="Italic">Suaeda glauca</Emphasis>

Bioassay-guided fractionation of the MeOH extract of Suaeda glauca yielded four phenolic compounds, methyl 3,5-di-O-caffeoyl quinate (1) and 3,5-di-O-caffeoyl quinic acid (2), isorhamnetin 3-O-beta-D-galactoside (3), and quercetin 3-O-beta-D-galactoside (4). Compounds 1 and 2 were hepatoprotective against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells with the EC(50) values of 72.7+/-6.2 and 117.2+/-10.5 microM, respectively. Silybin as a positive control showed an EC(50) value of 82.4+/-4.1 microM.     

MDMA induces <Emphasis Type="Italic">Per1</Emphasis>, <Emphasis Type="Italic">Per2</Emphasis> and <Emphasis Type="Italic">c-fos</Emphasis> gene expression in rat suprachiasmatic nuclei

Rationale  

±3,4-Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) is a psychoactive drug that has marked effects on the serotonergic system. Serotonergic agonists are known to interact with the circadian pacemaker located in the suprachiasmatic nuclei (SCN).