Tests with Daphnia magna: a new approach to prescreen toxicity of newly synthesized acetylcholinesterase reactivators

Reactivators of phosphorylated acetylcholinesterase (oximes) are substances used as a human antidotal therapy for organophosphate poisoning. The objective of our study was to examine if juveniles of freshwater microcrustacean Daphnia magna could be employed as test animals in early screen toxicity tests of those substances as a first step for further experiments with daphnids intoxicated by organophosphates. For this purpose, seven different oximes were investigated. It was found that toxicity of all tested oximes increased with time. Mono-quaternary oximes were approximately ten fold (EC50, 14.9 mg.l(-1)) more toxic in 24 hour tests and five fold (EC50 was 79.46 mg.l(-1)) more toxic in 48 hour tests than bis-quaternary oximes. Tests with daphnids were shown to be easy to carry out at low cost and provided valuable results which could be used as a starting point for further research.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.     

不同种类农药表面活性剂对大型溞的急性毒性

运用评价化学品对水生生物毒性的标准试验方法,比较了39种非离子型、6种阴离子型和3种阳离子型的常用代表性表面活性剂对大型溞的急性毒性.结果表明:3种阳离子型表面活性剂1427、1227及C8-10的急性毒性均为剧毒,其中1427毒性最高,EC50值为0.97×10-2 mg·L-1;非离子型表面活性剂中蓖麻油聚氧乙烯醚、吐温和斯潘系列乳化剂均为低毒,而烷基酚聚氧乙烯醚系列和脂肪醇聚氧乙烯醚系列表面活性剂的毒性稍偏高,而AEO-7和AEO-5的毒性达到高毒水平,EC50值分别为0.82和0.97 mg·L-1,且此类表面活性剂脂溶性越大,对大型溞的毒性越大;大部分阴离子表面活性剂的毒性为中毒,但NNO表现为高毒,EC50值为0.17 mg·L-1.     

A new approach to toxicity testing in Daphnia magna: application of high throughput FT-ICR mass spectrometry metabolomics

Currently there is a surge of interest in exploiting toxicogenomics to screen the toxicity of chemicals, enabling rapid and accurate categorisation into classes of defined mode-of-action (MOA), and prioritising chemicals for further testing. Direct infusion FT-ICR mass spectrometry-based metabolomics can provide a sensitive and unbiased analysis of metabolites in only 15 mins and therefore has considerable potential for chemical screening. The water flea, Daphnia magna, is an OECD test species and is utilised internationally for toxicity testing. However, no metabolomics studies of this species have been reported. Here we optimised and evaluated the effectiveness of FT-ICR mass spectrometry metabolomics for toxicity testing in D. magna. We confirmed that high-quality mass spectra can be recorded from as few as 30 neonates (<24 h old; 224 μg dry mass) or a single adult daphnid (301 μg dry mass). An OECD 24 h acute toxicity test was conducted with neonates at copper concentrations of 0, 5, 10, 25, 50 μg l⁻¹. A total of 5447 unique peaks were detected reproducibly, of which 4768 were assigned at least one empirical formula and 1017 were putatively identified based upon accurate mass measurements. Significant copper-induced changes to the daphnid metabolome, consistent with the documented MOA of copper, were detected thereby validating the approach. In addition, N-acetylspermidine was putatively identified as a novel biomarker of copper toxicity. Collectively, our results highlight the excellent sensitivity, reproducibility and mass accuracy of FT-ICR mass spectrometry, and provide strong evidence for its applicability to high-throughput screening of chemical toxicity in D. magna. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Toxicity of the new pyrethroid insecticide,deltamethrin, to Daphnia magna

The toxicity of deltamethrin, a synthetic pyrethroid insecticide, was determined under standardized conditions (ISO, 1982) in neonates and juveniles of Daphnia magna. Neonates (6 to 24 h old) were more sensitive than juveniles (48 to 72 h old). The 24- and 48-h EC₅₀s (immobilization) in neonates were 0.113 and 0.031 µg l^–1, respectively. The toxicity of deltamethrin was highly toxic. The 96-h EC₅₀ was in the ppt (µg l^–1) range. Toxicity tests with Daphnia may be used to detect toxic residues in water and sediment in areas treated with deltamethrin and other highly toxic pyrethroid pesticides.     

Effect of food concentration on the chronic toxicity of sodium dodecyl sulphate to Daphnia magna

Food concentration supplied during subchronic and chronic toxicity tests, is one of the most important factors that might influence the response of test organisms to toxicants. The green microalga Scenedesmus incrassatulus was used as food for the cladoceran Daphnia magna, in a chronic toxicity test with the toxicant sodium dodecyl sulphate (SDS). Test concentrations were 0.625, 1.25, and 2.5 mg l^-1 of SDS, equivalent to 1/40, 1/20, and 1/10 of the average 48-h LC50 previously determined. Food concentrations were 9.5, 19, and 38 mg l^-1 (dry wt.). Survival and reproduction were recorded, and the data were analyzed using a life-table approach. After 55 days, the main findings were as follows:

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H⁺ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88–95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of K₁ for edrophonium, decamethonium and Al³⁺.     

The use of biomarkers in Daphnia magna toxicity testing. IV. Cellular Energy Allocation: a new methodology to assess the energy budget of toxicant-stressed Daphnia populations

The Cellular Energy Allocation (CEA) methodology wasdeveloped as biomarker technique to assess the effectof toxic stress on the energy budget of testorganisms. This short-term assay is based on thebiochemical assessment of changes in the energyreserves (total carbohydrate, protein and lipidcontent) and the energy consumption (electrontransport activity). The CEA methodology was evaluatedusing Daphnia magna juveniles exposed for 96hto sublethal lindane and mercury chlorideconcentrations. The ecological relevance of the CEAassay was assessed by comparing the sub-organismalresponse with population level parameters (obtainedfrom 21 day life table experiments) such as theintrinsic rate of natural increase (r_m) and themean total offspring per female. Two differentmethodologies were used to assess the effect levels:the no (lowest) observed effect level (NOAECs-LOAECs)approach and the regression-based approach. Bothtoxicants caused a significant decrease in the netenergy budget of D. magna, with a LowestObserved (Adverse) Effect Concentration (LOAEC) of0.18 mg/l and 5.6 µg/l for lindane andHgCl₂,respectively. Changes in the lipid content of theorganisms were detected at toxicant concentrationslower than those affecting the total carbohydrate andprotein content. Toxicant specific effects wereobserved on the electron transport activity.Comparison of the CEA results with those of thepopulation level tests revealed that for mercury theCEA based LOAEC was a three times lower than thatbased on r_m and the total brood size(18 µg/l). For lindane the CEA based LOAEC was twotimes lower than the LOAEC based on r_m(0.32 mg/l) but was higher than that based on thetotal number of offspring produced (0.1 mg/l).Using the regression-based approach, EC₁₀ valueswere calculated using three parameter sigmoid orlogistic models. Comparison between the CEA andr_m based EC₁₀ values demonstrates that forboth chemicals similar effect concentrations areobtained: the CEA-based EC₁₀ (0.20 mg/l) forlindane is 1.5 times higher than the r_m-basedEC₁₀ threshold (0.13 mg/l), while for mercury thebiomarker-based EC₁₀ value (9 µg/l) was 1.4times lower than the population-based EC₁₀ value(12.5 µg/l).From these results, we suggest that the short-term CEAassay may be useful for predicting long-term effectsat the population level. The consequences of theobserved effects on the energy budget of the testorganism are discussed in the context of the effectsemerging at the population and community level.     

Testing the pluralist approach to sex: the influence of environment on synergistic interactions between mutation load and parasitism in Daphnia magna

Both deleterious mutations and parasites have been acknowledged as potential selective forces responsible for the evolutionary maintenance of sexual reproduction. The pluralist approach to sex proposes that these two factors may have to interact synergistically in order to stabilize sex, and one of the simplest ways this could occur is if parasites are capable of causing synergistic epistasis between mutations in their hosts. However, the effects of both deleterious mutations and parasitism are known to be influenced by a range of environmental factors, so the nature of the interaction may depend upon the organisms' environment. Using chemically mutated Daphnia magna lines, we examined the effects of mutation and parasitism under a range of temperature and food regimes. We found that although parasites were capable of causing synergistic epistasis between mutations in their hosts, these effects were dependent upon an interaction between parasite genotype and temperature.     

A new approach for testing contaminated marine sediments: fertilization success of lugworms following parental exposure

A sediment bioassay is being developed using several marine benthic invertebrates to assess the effects of parental transfer of contaminants to the gametes. In this preliminary study, the emphasis was placed on developing methods for the in vitro fertilization of lugworm, Arenicola marina, oocytes.Lugworms exposed to contaminated sediments in outdoor mesocosms were brought to the laboratory, just before the beginning of the spawning period. The reliability of an in vitro fertilization procedure was tested by varying several parts of the method. Main results are that eggs and embryos may be physically damaged by cleaning over a sieve. However, as no negative effects were observed when leaving eggs and sperm together for 24 h, the sperm need not be washed off until the embryos are preserved for further examination later on.A first, incomplete screening of the effects of contaminated harbour dredged sediments indicated some effect on the reproductive success.     

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.     

Resistance to boron toxicity amongst several barley and wheat cultivars: A preliminary examination of the resistance mechanism

The mechanism of resistance toB toxicity in barley and wheat was studied in a solution culture experiment using several cultivars displaying a large range of sensitivity to excessB supply. Plants were cultured for 35 d atB concentrations ranging from normal to excessive (15 to 5000 M, respectively) then examined for dry matter production and theB distribution between roots and shoots.In both species, increasedB supply was accompanied by increased tissueB concentrations, development ofB toxicity symptoms and depressed growth. At each level ofB supply, however, resistant cultivars accumulated considerably lessB than did sensitive cultivars, in both roots and shoots. Even at the lowestB supply, at which noB toxicity symptoms developed and growth was not affected, resistant cultivars maintained relatively low tissueB concentrations. No cultivar displayed an ability to tolerate high tissueB concentrations.These results indicate that sensitivity toB toxicity in barley and wheat is governed by the ability of cultivars to excludeB. If theB concentrations of tissues is used to indicate resistance toB toxicity, then cultivars have the same ranking whether cultured at a normal or excessB supply.     

Juliflorine: A potent natural peripheral anionic-site-binding inhibitor of acetylcholinesterase with calcium-channel blocking potential, a leading candidate for Alzheimer’s disease therapy

The alkaloid juliflorine (1) from Prosopis juliflora inhibited acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes in a concentration-dependent fashion with IC₅₀ values 0.42 and 0.12 μM, respectively. Lineweaver-Burk as well as Dixon plots and their secondary replots indicated that the nature of inhibition was purely of non-competitive type with K_i values 0.4 and 0.1 μM, against AChE and BChE, respectively. By molecular docking studies compound 1 was found to be ideally spaced inside the aromatic gorge of AChE with rings A/B remaining at the top and rings C/D penetrating deep into the gorge, that might be due to the greater hydrophobicity of rings C/D as compared to rings A/B, allowing their simultaneous interaction with the peripheral anionic and quaternary ammonium-binding sites. The 1-AChE complex was found to be stabilized by hydrophobic contacts, hydrogen bonding, and π-π stacking between the compound 1 and amino acid residues of the aromatic gorge of AChE. Amino acid residues Tyr70, Asp72, Tyr121, Trp279, and Tyr334 of the peripheral anionic site (PAS) of AChE were found to be exclusively involved in the hydrophobic contacts with compound 1 that might be responsible for the competitive mode of inhibition. Compound 1 also showed dose-dependent (30-500 μg/mL) spasmolytic and Ca²⁺-channel blocking activities in isolated rabbit jejunum preparations. The cholinesterase inhibitory potential along with calcium-channel blocking activity of compound 1 and safe profile in human neutrophils viable assay could make it a possible drug candidate for Alzheimer’s disease.     

A nonparametric approach to the analysis of longitudinal data via a set of level crossing problems with application to the analysis of microarray time course experiments

Here we develop a completely nonparametric method for comparing two groups on a set of longitudinal measurements. No assumptions are made about the form of the mean response function, the covariance structure or the distributional form of disturbances around the mean response function. The solution proposed here is based on the realization that every longitudinal data set can also be thought of as a collection of survival data sets where the events of interest are level crossings. The method for testing for differences in the longitudinal measurements then is as follows: for an arbitrarily large set of levels, for each subject determine the first time the subject has an upcrossing and a downcrossing for each level. For each level one then computes the log rank statistic and uses the maximum in absolute value of all these statistics as the test statistic. By permuting group labels we obtain a permutation test of the hypothesis that the joint distribution of the measurements over time does not depend on group membership. Simulations are performed to investigate the power and it is applied to the area that motivated the method-the analysis of microarrays. In this area small sample sizes, few time points and far too many genes to consider genuine gene level longitudinal modeling have created a need for a simple, model free test to screen for interesting features in the data.     

A new theoretical approach to the investigation of the symmetry of protein oligomers with bifunctional reagents

The use of bifunctional reagents to form cross-links between subunits in protein oligomers and subsequent disruption of noncovalent interactions with SDS allows comment upon the number of subunits and the symmetry in the original assembly. In existing treatments the number of equations needed to describe theoretically the proportions of all the cross-linked species that can be formed as a function of time in this way makes the analysis of the system unmanageable for proteins with more than four subunits. A method is presented that allows the required equations for any oligomer to be formulated as an algorithm suitable for solution by computer. Its application is illustrated with reference to experimental results obtained with two protein hexamers, Jasus hemocyanin and alpha-urease from jack bean.     

A new approach to the determination of cardiac potential distributions: application to the analysis of electrode configurations

This paper presents a mathematical model and new solution technique for studying the electric potential in a slab of cardiac tissue. The model is based on the bidomain representation of cardiac tissue and also allows for the effects of fibre rotation between the epicardium and the endocardium. A detailed solution method, based on Fourier Series and a simple one-dimensional finite difference scheme, for the governing equations for electric potential in the tissue and the blood, is also presented. This method has the advantage that the potential can be calculated only at points where it is required, such as the measuring electrodes. The model is then used to study various electrode configurations which have been proposed to determine cardiac tissue conductivity parameters. Three electrode configurations are analysed in terms of electrode spacing, placement position and the effect of including fibre rotation: the usual surface four-electrode configuration; a single vertical analogue of this and a two probe configuration, which has the current electrodes on one probe and the measuring electrodes on the other, a fixed distance away. It is found that including fibre rotation has no effect on the potentials measured in the first two cases; however, in the two probe case, non-zero fibre rotation causes a significant drop in the voltage measured. This leads to the conclusion that it is necessary to include the effects of fibre rotation in any model which involves the use of multiple plunge electrodes.     

A new approach for quantitative evaluation of cross-reactivity of steroids with an antiserum by radioimmunoassay: application to a highly specific antiestriol

The study of several antiestriol antisera in the presence of a series of analogues of estriol has led to a critical discussion of the classical cross-reaction test. A more accurate test (CR₁ ng) which permits a more precise determination of specificity of antisera is described.     

Reduction of oxidative stress induced vanadium toxicity by complexing with a flavonoid, quercetin: A pragmatic therapeutic approach for diabetes

Vanadium compounds are known to lower blood glucose level in diabetes but are associated with toxicity. In vitro cytotoxicity of VOSO₄ and bis(quercetinato) oxovanadium(IV) (BQOV) was examined in CHO cells. Both the agents showed time and dose dependent increase in ROS generation however it was relatively less in BQOV. Moreover, VOSO₄ also caused higher necrosis. Hypoglycemic potential of VOSO₄ and BQOV was tested in streptozotocin-induced diabetic Balb/c mice. A marked difference was observed in the hypoglycemic action of VOSO₄ and BQOV treated mice that lasted only for about 6 h in VOSO₄ as against 24 h in BQOV. Comparison of acute toxicity of the compounds in normal Balb/c mice revealed negligible nephrotoxicity of BQOV. Kidney analyses of VOSO₄ treated animals’ revealed high ROS generation and tubular necrosis. Similarly serum levels of urea and creatinine were elevated in these animals indicating kidney dysfunction. No such abnormality was observed in BQOV treated animals. Reduced nephrotoxicity of BQOV could be due to increased catalase activity found in the kidney of BQOV treated animals and BQOV’s radical scavenging activity. The data clearly demonstrates immense hypoglycemic activity and reduced toxicity of BQOV thus making the conjugate a suitable candidate for therapeutic utility.