Ingestion and contact with polyethylene microplastics does not cause acute toxicity on marine zooplankton.

Toxicity of polyethylene microplastics (PE-MP) of size ranges similar to their natural food to zooplanktonic organisms representative of the main taxa present in marine plankton, including rotifers, copepods, bivalves, echinoderms and fish, was evaluated. Early life stages (ELS) were prioritized as testing models in order to maximize sensitivity. Treatments included particles spiked with benzophenone-3 (BP-3), a hydrophobic organic chemical used in cosmetics with direct input in coastal areas. Despite documented ingestion of both virgin and BP-3 spiked microplastics no acute toxicity was found at loads orders of magnitude above environmentally relevant concentrations on any of the invertebrate models. In fish tests some effects, including premature or reduced hatching, were observed after 12 d exposure at 10 mg L-1 of BP-3 spiked PE-MP. The results obtained do not support environmentally relevant risk of microplastics on marine zooplankton. Similar approaches testing more hydrophobic chemicals with higher acute toxicity are needed before these conclusions could be extended to other organic pollutants common in marine ecosystems. Therefore, the replacement of these polymers in consumer products must be carefully considered.

An ecotoxicological view on neurotoxicity assessment.

The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.

Trophic contamination by pyrolytic polycyclic aromatic hydrocarbons does not affect aerobic metabolic scope in zebrafish Danio rerio.

The effect of trophic exposure to pyrolitic polycyclic aromatic hydrocarbons (PAH) on aerobic metabolism of zebrafish Danio rerio was investigated. There were no significant differences in standard metabolic rate (SMR), active metabolic rate (AMR) or aerobic metabolic scope (AS) at any sublethal concentration of PAH in the diet of adult or juvenile fish. This suggests that under these experimental conditions, exposure to PAH in food did not influence aerobic metabolism of this species.

Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenic but not genotoxic effects.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be present at high levels as mixtures in polluted aquatic environments. Many PAHs are potent mutagens and several are well-known carcinogens. Despite numerous studies on individual compounds, little is known about the toxicity of PAHs mixtures that are encountered in environmental situations. In the present work, zebrafish were continuously fed from 5 days post-fertilisation to 14 months post-fertilisation (mpf) with a diet spiked with fractions of either pyrolytic (PY), petrogenic light oil (LO), or petrogenic heavy oil (HO) origin at three concentrations. A decrease in survival was identified after 3 mpf in fish fed with the highest concentration of HO or LO, but not for PY. All PAH fractions caused preneoplastic and neoplastic disorders in long-term-exposed animals. Target tissues were almost exclusively of epithelial origin, with the bile duct epithelium being the most susceptible to chronic exposure to all PAH fractions, and with germ cells being the second most responsive cells. Significantly higher incidences of neoplasms were observed with increasing PAH concentration and exposure duration. The most severe carcinogenic effects were induced by dietary exposure to HO compared to exposure to LO or PY (45, 30 and 7 %, respectively, after 9 to 10 months of exposure to an intermediate concentration of PAHs). In contrast, earliest carcinogenic effects were detected as soon as 3 mpf after exposure to LO, including the lowest concentration, or to PY. PAH bioactivation and genotoxicity in blood was assessed by ethoxyresorufin-O-deethylase activity quantification and comet and micronuclei assays, respectively, but none of these were positive. Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenotoxic events that impair survival and physiology of exposed fish.

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio.

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R(A)) and standard (R(S)) metabolic rate at different life stages in zebrafish Danio rerio (5 day-old larvae, 2 month-old juveniles and 6 month-old adults). Scaling exponents and constants were assessed for standard (R(S) = 0·273M(0·965) in mgO(2) g(-1) h(-1)) and active metabolic rate (R(A) = 0·799M(0·926) in mgO(2) g(-1) h(-1)). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.

Links between kinetic data and sequences in the alpha/beta-hydrolases fold database.

While the number of sequenced genes is increasing dramatically, the number of different protein structural families is expected to be more limited. Changes in enzymatic activity or protein interactions can dramatically modify the role of homologous proteins in different organisms or mutants. However, experimental data associated with sequences or mutations stored in databases are often limited to a short description of the enzymatic pathway, molecular interaction or phenotype associated with the changes in amino acid sequence. In the alpha/beta-hydrolases fold database ESTHER, we are experimenting with links between experimental kinetic data and sequences, mutations and protein structures. This effort will lead to the integration of pharmacological data with genome-wide databases.

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface.

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

Acetylcholinesterase from snake venom as a model for its nerve and muscle counterpart.

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. At the neuromuscular junction of vertebrates, for example, it allows a fine temporal control of muscle contraction. The presence of AChE in tissues devoid of cholinergic function is also well known and raises the question of its role. In particular, AChE is abundant in the venoms of Elapid snakes, except mambas. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning of cDNA of the AChE from Bungarus fasciatus venom showed that its C-terminal peptide is very different from those of other AChEs. The partial sequence of the Bungarus fasciatus AChE gene shows that this peptide is encoded by a new alternative exon, called S for soluble and snake. It is a short very basic peptide of 15 residues. Analysis of the venom enzyme and in vitro expression experiments showed that the last eight residues are removed in the mature protein. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to fasciculins from mamba venoms. While Ophiophagus AChE is as sensitive as Torpedo enzyme (IC50 around 10(-10) M), Naja and Heamacatus AChEs are insensitive to the toxin up to a concentration of 10(-6) M Bungarus AChE has an intermediate IC50 of 10(-8) M. Analysis of its sequence reveals two major differences in the peripheral site region, compared to Torpedo or mammalian AChEs: at position 70 it contains a methionine instead of a tyrosine, and at position 285 it contains a lysine instead of an acidic residue (glutamic or aspartic acid). Modification of these residues by site-directed mutagenesis, and enzymatic analysis of modified recombinant enzymes, confirmed that these two residues are implicated in the properties of the Bungarus AChE peripheral site. The presence of an alternative exon, which generates a soluble form of AChE in venoms, raises interesting evolutionary questions: Does it exist in snakes whose venom does not contain AChE, e.g., mambas? Did this exon pre-exist, for expression in other contexts? Snake venom AChEs offer an exceptional system for analyzing the mechanism of peripheral site inhibition, because of their wide range of activities.

Kinetic parameters of cholinesterase interactions with organophosphates: retrieval and comparison tools available through ESTHER database: ESTerases, alpha/beta Hydrolase Enzymes and Relatives.

Cholinesterases are targets for organophosphorus compounds which are used as insecticides, chemical warfare agents and drugs for the treatment of disease such as glaucoma, or parasitic infections. The widespread use of these chemicals explains the growing of this area of research and the ever increasing number of sequences, structures, or biochemical data available. Future advances will depend upon effective management of existing information as well as upon creation of new knowledge. The ESTHER database goal is to facilitate retrieval and comparison of data about structure and function of proteins presenting the alpha/beta hydrolase fold. Protein engineering and in vitro production of enzymes allow direct comparison of biochemical parameters. Kinetic parameters of enzymatic reactions are now included in the database. These parameters can be searched and compared with a table construction tool. ESTHER can be reached through internet (http://www.ensam.inra.fr/cholinesterase). The full database or the specialised X-window Client-server system can be downloaded from our ftp server (ftp://ftp.toulouse.inra.fr./pub/esther). Forms can be used to send updates or corrections directly from the web.

Production of an immunoenzymatic tracer combining a scFv and the acetylcholinesterase of Bungarus fasciatus by genetic recombination.

We constructed a plasmid containing a chimeric gene composed of the gene encoding acetylcholinesterase (AChE) from Bungarus fasciatus venom and a gene encoding a single chain antibody fragment (scFv) directed against one of the two subunits of a presynaptic neurotoxin from rattlesnake. Large quantities of the fusion protein were produced in the culture medium of transfected COS cells. Fusion to AChE did not affect the ability of the scFv to recognise its antigen. Similarly, the AChE activity was not impaired in the fusion. The fusion protein was purified from the culture medium in a single step by affinity chromatography. The immunoconjugate obtained consisted of a soluble monomeric form of AChE fused to scFv. It was monovalent and had a molecular weight of 94 kDa. The properties of this scFv-AChE fusion show that the simple, reproducible preparation of various recombinant monovalent immunoenzymatic tracers with low molecular weight is possible. In addition, in the construct presented, the scFv domain can be easily changed to another one taking advantage of the SfiI-NotI restriction sites surrounding this domain.

Identification of a novel type of alternatively spliced exon from the acetylcholinesterase gene of Bungarus fasciatus. Molecular forms of acetylcholinesterase in the snake liver and muscle.

The venom of the snake Bungarus fasciatus contains a hydrophilic, monomeric species of acetylcholinesterase (AChE), characterized by a C-terminal region that does not resemble the alternative T- or H-peptides. Here, we show that the snake contains a single gene for AChE, possessing a novel alternative exon (S) that encodes the C-terminal region of the venom enzyme, located downstream of the T exon. Alternative splicing generates S mRNA in the venom gland and S and T mRNAs in muscle and liver. We found no evidence for the presence of an H exon between the last common "catalytic" exon and the T exon, where H exons are located in Torpedo and in mammals. Moreover, COS cells that were transfected with AChE expression vectors containing the T exon with or without the preceding genomic region produced exclusively AChET subunits. In the snake tissues, we could not detect any glycophosphatidylinositol-anchored AChE form that would have derived from H subunits. In the liver, the cholinesterase activity comprises both AChE and butyrylcholinesterase components; butyrylcholinesterase corresponds essentially to nonamphiphilic tetramers and AChE to nonamphiphilic monomers (G1na). In muscle, AChE is largely predominant: it consists of globular forms (G1a and G4a) and trace amounts of asymmetric forms (A8 and A12), which derive from AChET subunits. Thus, the Bungarus AChE gene possesses alternatively spliced T and S exons but no H exon; the absence of an H exon may be a common feature of AChE genes in reptiles and birds.

aCHEdb: the database system for ESTHER, the alpha/beta fold family of proteins and the Cholinesterase gene server.

Acetylcholinesterase belongs to a family of proteins, the alpha/beta hydrolase fold family, whose constituents evolutionarily diverged from a common ancestor and share a similar structure of a central beta sheet surrounded by alpha helices. These proteins fulfil a wide range of physiological functions (hydrolases, adhesion molecules, hormone precursors) [Krejci,E., Duval,N., Chatonnet,A., Vincens,P. and Massoulié,J. (1991) Proc. Natl. Acad. Sci. USA , 88, 6647-6651]. ESTHER (for esterases, alpha/beta hydrolase enzymes and relatives) is a database aimed at collecting in one information system, sequence data together with biological annotations and experimental biochemical results related to the structure-function analysis of the enzymes of the family. The major upgrade of the database comes from the use of a new database management system: aCHEdb which uses the ACeDB program designed by Richard Durbin and Jean Thierry-Mieg. It can be found at http://www.ensam.inra.fr/cholinesterase

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Neurobehavioral evaluation of lurcher mutant mice during ontogeny.

Lurcher mutant mice were compared to normal littermate controls for body weight, body righting, negative geotropism, sensorimotor coordination (rotating grid, wire suspension, rotorod), and visuomotor coordination requiring swimming toward a pole during postnatal (P) days 0-30. Lurcher mutants had a lower body weight on P20-P30 and were slower before performing the complete body righting response on P13-P30. Because of postural instability during the negative geotropism test, lurcher mutants turned quicker up the slope than normal mice. The mutants fell sooner from the rotating grid on P11-P14, from the horizontal wire on P15-P16, and from the rotorod on P14-P30. Lurcher mutants were also slower before swimming to the pole or climbing to the top of the pole and were inferior in pole climbing height on P22-P30. These results indicate test-selective and time-selective neurobehavioral deficits during ontogeny in a spontaneous cerebellar mutant.

The alpha/beta fold family of proteins database and the cholinesterase gene server ESTHER.

ESTHER (for esterases, alpha/betahydrolase enzyme and relatives) is a database of sequences phylogenetically related to cholinesterases. These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) sharing a similar structure of a central beta-sheet surrounded by alpha-helices. Among these proteins a wide range of functions can be found (hydrolases, adhesion molecules, hormone precursors). The purpose of ESTHER is to help comparison of structures and functions of members of the family. Since the last release, new features have been added to the server. A BLAST comparison tool allows sequence homology searches within the database sequences. New sections are available: kinetics and inhibitors of cholinesterases, fasciculin-acetylcholinesterase interaction and a gene structure review. The mutation analysis compilation has been improved with three-dimensional images. A mailing list has been created.

[Acetylcholinesterase from snake venoms]. / L'acétylcholinestérase des venins de serpents.

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. For example, located at the neuro-muscular junction of vertebrates, it allows a fine temporal control of muscle contraction. The presence of AChE in tissues devoid of cholinergic function is also well known and raises the question of its role. In particular, AChE occurs at high level in the venoms of Elapid snakes, except Mambas. In contrast, the venom of snakes belonging to Viperid or Colubrid families does not contain any AChE. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning the cDNA of the venom AChE from Bungarus fasciatus showed that its C-terminal peptide is very different from those of other AChEs. This peptide is encoded by a new alternative exon, called S for Soluble and Snake. It is a short very basic peptide of 15 residues. Analysis of the venom enzyme and in vitro expression experiments showed that the last eight residues are removed in the mature protein. This cleavage is not necessary for enzymatic activity and occurs before secretion of the enzyme. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to Mambas toxins, fasciculins. While Ophiophagus AChE is as sensitive as Torpedo enzyme (IC50 around 10(-10) M), Naja and Heamacatus AChEs are insensitive to the toxin up to a concentration of 10(-6) M. Bungarus AChE has an intermediary IC50 of 10(-8) M. The analysis of its sequence shows two major differences, in the peripheral site region, when compared to Torpedo or mammalian AChEs: at position 70 it contains a methionine instead of a tyrosine and at position 285, it contains a lysine instead of an acidic residue (glutamic or aspartic acid). The modification of these residues by site-directed mutagenesis and the enzymatic analysis of modified recombinant enzymes confirmed the implication of these two residues in the properties of Bungarus AChE peripheral site. The presence of an alternative exon, used for generating a soluble form of AChE in venoms, raises interesting evolutionary questions: does it exist in snakes whose venom does not contain AChE, e.g. Mambas? did this exon preexist, for expression in other contexts? In addition snake venoms offer an exceptional system for analysing the mechanism of peripheral site inhibition, because of its wide range of sensitivity.

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non-amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N-glycans of the 'complex' and 'hybrid' types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non-denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

Cloning and expression of acetylcholinesterase from Bungarus fasciatus venom. A new type of cooh-terminal domain; involvement of a positively charged residue in the peripheral site.

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.