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.

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.

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.

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.

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.

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.

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.

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.

cDNA sequence, gene structure, and cholinesterase-like domains of an esterase from Caenorhabditis elegans mapped to chromosome V.

The structure of an esterase gene from Caenorhabditis elegans has been determined by comparison of the sequences in genomic and cDNA clones. The gene was mapped close to the center of chromosome V (1.7 centimorgans to the left of dpy-11) and is therefore distinct from the gut esterase gene ges-1. It possessed 7 short introns. The 5' splice site of intron 3 presented the sequence GC instead of the usual GT that was found in the other six introns. The cDNA was trans-spliced with the short leader SL1. The open reading frame indicated that a protein of 557 aminoacids was encoded. The deduced aminoacid sequence did not present a signal peptide at the N-terminal but a potential N-myristoylation site (GXXXS) provided that the initiator methionine was removed. This protein should therefore remain intracellular. Comparison of this C. elegans sequence to other protein sequences in databases, as well as the analysis of the secondary structure in the protein showed that it belongs to the subgroup of esterases in the alpha/beta hydrolase fold family.

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.

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.

[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.

Adenylate cyclase in the developing rat cerebral cortex and olfactory bulb.

Activities of adenylate cyclase, measured either in the absence or presence of sodium fluoride and Triton X-100, are determined in cerebral cortex and olfactory bulb homogenate of rats of 1 to 35 days of postnatal age. Differences in properties of the enzyme in the 2 structures are demonstrated.

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.

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.

A cholinesterase genes server (ESTHER): a database of cholinesterase-related sequences for multiple alignments, phylogenetic relationships, mutations and structural data retrieval.

We have built a database of sequences phylogenetically related to cholinesterases (ESTHER) for esterases, alpha/beta hydrolase enzymes and relatives). These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) with some related proteins devoid of enzymatic activity. The purpose of ESTHER is to help comparison and alignment of any new sequence appearing in the field, to favour mutation analysis of structure-function relationships and to allow structural data recovery. ESTHER is a World Wide Web server with the URL http://www.montpellier.inra.fr:70/cholinesterase.

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.