Inhibition with simultaneous spontaneous reactivation and aging of acetylcholinesterase by organophosphorus compounds: Demeton-S-methyl as a model.

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus, carbamates and sulfonylfluorides) sometimes cannot yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work kinetic data were obtained for demeton-S-methyl (DSM) with human acetylcholinesterase in two kinds of experiments: (a) time progressive inhibition with a range of concentrations, (b) progressive spontaneous reactivation starting with pre-inhibited enzyme. DSM is an organophosphorus compound used as pesticide and considered a model for studying the dermal exposure of nerve agents such as VX gas. A kinetic model equation was deduced with four different molecular phenomena occurring simultaneously: (1) inhibition; (2) spontaneous reactivation; (3) aging; and (4) ongoing inhibition (inhibition during the substrate reaction). A 3D fit of the model was applied to analyze the inhibition experimental data. The best-fitting model is compatible with a sensitive enzymatic entity. The second-order rate constant of inhibition (ki = 0.0422 µM-1 min-1), the spontaneous reactivation constant (ks = 0.0202 min-1) and the aging constant (kg = 0.0043 min-1) were simultaneously estimated. As an example for testing the model and approach, it was tested also in the presence of 5 % ethanol (conditions as previously used in the literature), the best fitting model is compatible with two apparent sensitive enzymatic entities (17 % and 83 %) and only one spontaneously reactivates and ages. The corresponding second-order rate constants of inhibition (ki = 0.0354 and 0.0119 µM-1 min-1) and the spontaneous reactivation and aging constants for the less sensitive component (kr = 0.0203 min-1 and kg = 0.0088 min-1) were estimated. The results were also consistent with a significant ongoing inhibition. These parameters were similar to those deduced in spontaneous reactivation experiments of the pre-inhibited samples with DSM in the absence or presence of ethanol. The two apparent components fit was interpreted by an equilibrium between ethanol-free and ethanol-bound enzyme. The consistency of results in inhibition and in spontaneous reactivation experiments was considered an internal validation of the methodology and the conclusions.

Investigation of the Effects of Metallic Nanoparticles on Fertility Outcomes and Endocrine Modification of the Hypothalamic-Pituitary-Gonadal Axis.

Nanotechnology is a very disruptive twenty-first-century revolution that will allow social and economic welfare to increase although it also involves a significant human exposure to nanoparticles. The aim of the present study was to contribute to the elucidation on whether metallic nanoparticles have a potential to induce fertility impairments. Regulatory studies that observed official OECD guidelines 415, 416 and 422 have failed to detect any fertility alterations caused by nanoparticle exposure. However, the scientific literature provides evidence that some nanoparticles may cause gonad impairments although the actual impact on fertility remains uncertain. This aim of the present study is to revisit the previously published RNAseq studies by analyzing the effects of several nanoparticles on the transcriptome of T98G human glioblastoma cells given that glial cells are known to play a pivotal role in the regulation of gonadotropin releasing hormone neurons. We found evidence that nanoparticles impair the gonadotropin releasing hormone receptor pathway and several related biological process like, among others, the cellular response to follicular stimulating hormone, cellular response to gonadotropin stimulus, cellular response to hormone stimulus, response to steroid hormone, ovulation cycle and response to estradiol. We propose that nanoparticles interfere with the ability of glial cells to regulate gonadotropin-releasing hormone neurons and, subsequently, the hypothalamic-pituitary-gonadal axis, potentially leading to fertility impairments. To our knowledge, this is the first proposal of a mode of action based on endocrine disruption for explaining the possible effects of nanoparticles on fertility. Whether these finding can be extended to other types of nanoparticles requires further investigation.

Geological context and human exposures to element mixtures in mining and agricultural settings in Colombia.

Anthropogenic and natural sources contribute to chemical mixtures in air, water, and soil, posing potential risks to the environment and human health. To understand the interplay between element profiles in the human body, geographical location, and associated economic activities, we carried out an observational analytic cross-sectional study. The study recruited 199 participants from three municipalities, two of which had gold-mining as their primary economic activity, while the other was dedicated to agricultural and other local activities not related to mining. The concentrations of a total of 30 elements in human hair samples and 21 elements in environmental soil samples were measured using various spectrometry techniques. Unsupervised clustering analysis using Self-Organizing Maps was applied to human hair samples to analyze element concentrations. Distinct clusters of individuals were identified based on their hair element profiles, which were mapped to geographical location and economic activities. While higher levels of heavy metals (Ag, As, Hg, and Pb) were observed in individuals engaged in mining activities in certain clusters, individuals in agricultural areas show higher concentrations of elements found in pesticides (Ba and Sr). However, the elemental composition of hair is influenced not only by the anthropogenic activities but also by the inherent geological context where people live. Our findings highlight the significance of accounting for environmental factors when evaluating human health risks, as the intricate mixture of elements can yield valuable insights for targeted health interventions.

Reference Values on Children's Hair for 28 Elements (Heavy Metals and Essential Elements) Based on a Pilot Study in a Representative Non-Contaminated Local Area.

Studies have been published, and laboratories offer services of measuring elements in hair as biomarkers of environmental exposure and/or control of essential elements (trace or macro). These reported values can have only sense if compared with adopted reference values. In this work, we propose provisional reference values based on a pilot child population. The concentrations of 28 elements were measured in children's hair samples. An observational, descriptive, cross-sectional study was conducted in a typical child population in the Mediterranean region void of excessive pollution problems to analyze 419 hair samples of children aged 3-12 years. Children were selected by a simple random method from eight primary education schools in different municipal districts, which included urban, rural and industrial areas. Samples of around 100 mg were washed and acid digested by an optimized procedure. All measures were performed using ICP-MS with Sc, Y and Re as internal standards. The statistical analysis was performed by two approaches: (a) considering all the data and (b) without outliers (second-order atypical data) to compare them with other published studies. The distribution curves in all the elements studied were asymmetric and did not fit the theoretical normality distributions. Therefore, the analysis based on percentiles was more appropriate. In most elements, only slight differences were observed with sex or age, which did not justify proposing separate reference ranges. From the results of this study, provisional reference values are proposed following two criteria: (a) simple application of the table of percentiles built by removing outlier values and (b) values after a detailed analysis case-by-case, considering other data as the distribution profile and other published data of each element. Although the pilot sample was from a limited area, it was carefully selected to be representative of a general non-contaminated population. With this limitation, the proposed reference values might be useful for researchers and physicians until a wider geographical study is available for a large number of elements.

Chemical Element Mixtures and Kidney Function in Mining and Non-Mining Settings in Northern Colombia.

The exposure to chemical mixtures is a problem of concern in developing countries and it is well known that the kidney is the major target organ for toxic elements. This cross-sectional study aimed to estimate the individual and composite mixture effect of a large number of chemical elements on kidney function in gold-mining and surrounding non-mining populations in northeast Colombia. We measured concentrations of 36 chemical elements in hair as indicators of chronic exposure from 199 adult participants. We estimated the effect of exposure to mixtures of chemical elements on estimated glomerular filtration rate (eGFR) using weighted quantile sum regression (WQS). The WQS index of the mixture was associated with reduced eGFR (Coefficient -2.42; 95%CI: -4.69, -0.16) being Be, Cd, Pb, As, and Mn, the principal contributors of the toxic mixture. Mining activities and Hg concentration were not associated with decreased kidney function. Our results suggest that complex mixtures of chemical elements, mainly heavy metals, act as nephrotoxic in these populations and therefore the analysis of chemical element mixtures is a better approach to identify environmental and occupational chemical risks for kidney damage.

A Transcriptomic Analysis of T98G Human Glioblastoma Cells after Exposure to Cadmium-Selenium Quantum Dots Mainly Reveals Alterations in Neuroinflammation Processes and Hypothalamus Regulation.

Quantum dots are nanoparticles with very promising biomedical applications. However, before these applications can be authorized, a complete toxicological assessment of quantum dots toxicity is needed. This work studied the effects of cadmium-selenium quantum dots on the transcriptome of T98G human glioblastoma cells. It was found that 72-h exposure to 40 µg/mL (a dose that reduces cell viability by less than 10%) alters the transcriptome of these cells in biological processes and molecular pathways, which address mainly neuroinflammation and hormonal control of hypothalamus via the gonadotropin-releasing hormone receptor. The biological significance of neuroinflammation alterations is still to be determined because, unlike studies performed with other nanomaterials, the expression of the genes encoding pro-inflammatory interleukins is down-regulated rather than up-regulated. The hormonal control alterations of the hypothalamus pose a new concern about a potential adverse effect of quantum dots on fertility. In any case, more studies are needed to clarify the biological relevance of these findings, and especially to assess the real risk of toxicity derived from quantum dots exposure appearing in physiologically relevant scenarios.

Case study: risk associated to wearing silver or graphene nanoparticle-coated facemasks for protection against COVID-19.

The world is living a pandemic situation derived from the worldwide spreading of SARS-CoV-2 virus causing COVID-19. Facemasks have proven to be one of the most effective prophylactic measures to avoid the infection that has made that wearing of facemasks has become mandatory in most of the developed countries. Silver and graphene nanoparticles have proven to have antimicrobial properties and are used as coating of these facemasks to increase the effectivity of the textile fibres. In the case of silver nanoparticles, we have estimated that in a real scenario the systemic (internal) exposure derived from wearing these silver nanoparticle facemasks would be between 7.0 × 10-5 and 2.8 × 10-4 mg/kg bw/day. In addition, we estimated conservative systemic no effect levels between 0.075 and 0.01 mg/kg bw/day. Therefore, we estimate that the chronic exposure to silver nanoparticles derived form facemasks wearing is safe. In the case of graphene, we detected important gaps in the database, especially regarding toxicokinetics, which prevents the derivation of a systemic no effect level. Nevertheless, the qualitative approach suggests that the risk of dermal repeated exposure to graphene is very low, or even negligible. We estimated that for both nanomaterials, the risk of skin sensitisation and genotoxicity is also negligible.

Interactions of human acetylcholinesterase with phenyl valerate and acetylthiocholine: Thiocholine as an enhancer of phenyl valerate esterase activity.

Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site ("PVsite") were deduced.In this paper, we demonstrate that acetylcholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among substrates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems.

DAEH N-terminal sequence of avian serum albumins as catalytic center of Cu (II)-dependent organophosphorus hydrolyzing A-esterase activity.

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy. The R (+)-HDCP inhibits and caused the so call "aging reaction" on inhibited-NTE. This enantiomer is not hydrolyzed by Ca(II)-dependent A-esterases in mammal tissues but is hydrolyzed by Cu(II)-dependent chicken serum albumin (CSA). With the aim of identifying HDCP hydrolysis by other vertebrate albumins, we incubated albumin with 400 µM racemic HDCP in the presence of 100 µM copper sulfate. HDCPase activity was assessed by measurement of HDCP with chiral chromatography. Human, sheep, dog, pig, lamprey or cobra serum albumin did not show a significant activity (~10%). Rabbit and bovine albumins hydrolyzed both enantiomers of HDCP (25% and 50% respectively). Turkey serum albumin had more HDCPase activity (~80 µM remaining) than the chicken albumin (~150 µM remaining). No animal albumins other than chicken showed stereoselective hydrolysis. Preincubation of chicken albumin with 1 mM the histidine modifying agents, 100 µM N-bromosuccinimide (NBS) and Zn(II), inhibited its Cu(II)-dependent R (+)-HDCPase activity, where as other mM amino acids modifiers had no inhibitory effects. . These results confirm that the stereoselective hydrolysis of (+)-HDCP is a specific A-esterase catalytic property of chicken albumin. The higher HDCPase activity by turkey albumin suggests the amino-terminal sequence of avian albumins (DAEHK) is the active center of this Cu(II)-dependent A-esterase activity.

Titanium Dioxide, but Not Zinc Oxide, Nanoparticles Cause Severe Transcriptomic Alterations in T98G Human Glioblastoma Cells.

Titanium dioxide and zinc oxide are two of the most widely used nanomaterials. We assessed the effects of noncytotoxic doses of both nanomaterials on T98G human glioblastoma cells by omic approaches. Surprisingly, no effects on the transcriptome of T98G cells was detected after exposure to 5 µg/mL of zinc oxide nanoparticles during 72 h. Conversely, the transcriptome of the cells exposed to 20 µg/mL of titanium dioxide nanoparticles during 72 h revealed alterations in lots of biological processes and molecular pathways. Alterations to the transcriptome suggests that exposure to titanium dioxide nanoparticles might, potentially, compromise the integrity of the blood brain barrier integrity and cause neuroinflammation. The latter issue was further confirmed phenotypically with a proteomic analysis and by recording the release of interleukin 8. Titanium dioxide also caused autophagy, which was demonstrated through the increase in the expression of the autophagy-related 3 and microtubule associated protein 1 light chain 3 alpha genes. The proteomic analysis revealed that titanium dioxide nanoparticles might have anticancerigen properties by downregulating genes involved in the detoxication of anthracyclines. A risk assessment resulting from titanium dioxide exposure, focusing on the central nervous system as a potential target of toxicity, is necessary.

Effects of silver nanoparticles on T98G human glioblastoma cells.

Nanotechnology has been well developed in recent decades because it provides social progress and welfare. Consequently, exposure of population is increasing and further increases in the near future are forecasted. Therefore, assessing the safety of applications involving nanoparticles is strongly advisable. We assessed the effects of silver nanoparticles at a non-cytotoxic concentration on the performance of T98G human glioblastoma cells mainly by an omic approach. We found that silver nanoparticles are able to alter several molecular pathways related to inflammation. Cellular repair and regeneration were also affected by alterations to the fibroblast growth factor pathways operating mainly via mitogen-activated protein kinase cascades. It was concluded that, given the relevant role of glia on central nervous system maintenance homeostasis, exposure to silver nanoparticles could eventually lead to severe toxicity in the central nervous system, although current exposure levels do not pose a significant risk.

O-hexyl O-2,5-dichlorophenyl phosphoramidate as a substrate for domestic and sea bird serum A-esterases: Hydrolysis levels, Cu2+- and Zn2+-dependence and stereoselectivity.

O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy in hens. It has been used as a tool to identify new A-esterase activities in animal tissues. This study shows the EDTA-resistant, Cu2+- and Zn2+-dependent hydrolysis of racemic HDCP in domestic and sea bird serum using UV/Vis spectrophotometry and chiral chromatography. The results clearly show a significant (p ˂ 0.05) Cu2+- and Zn2+-dependent HDCP hydrolysis in the serum of all bird species versus EDTA, except for the Zn2+-dependent HDCPase activity from Yucatecan quail serum. The ratio of Cu2+/Zn2+ hydrolysis varied between 1 and 7 (intraspecies) and 15.6 (interspecies). EDTA affected the Cu2+- and Zn2+-dependent HDCPase activity in the range of 37-95% and 40-50%, respectively. HDCP hydrolysis activated by Cu2+ was significantly (p ˂ 0.05) stereoselective (R-(+)-HDCP ˃ S-(-)-HDCP) in chicken and sea bird serum. Its R-(+)-HDCP/S-(-)-HDCP ratios were 6.8 and 1.6-2.8, respectively. EDTA-resistant and zinc-dependent HDCP hydrolysis were not stereospecific in all bird sera tested. The present ex vivo study reinforces the idea that bird sera have HDCPase activity that is sensitive to divalent metals, resistant to EDTA and possibly associated with the protein albumin.

Case study: Is bisphenol S safer than bisphenol A in thermal papers?

The Risk Assessment Committee of the European Chemical Agency released a scientific opinion alerting that the risk associated with dermal occupational exposure to bisphenol A (BPA) via thermal paper might not be adequately controlled because the estimated exposure was around twice the Derived No Effect Level (DNEL) and the European Commission will effectively restrict BPA in thermal paper as soon as 2020. Bisphenol S (BPS) is currently being used as a BPA surrogate and is already widespread in thermal paper receipts. Based on publically available information in the scientific literature, we assessed the risk associated with dermal BPS exposure via thermal paper for the general and occupational populations to compare with BPA situation. We developed two exposure scenarios; one based on the total excreted BPS and another on exposure estimations by transferring BPS from the thermal paper matrix to skin. Both scenarios yielded similar exposures for the general population (0.016-0.013 µg/kg bw/day), but the exposure estimated for the workers in the second scenario (0.96 µg/kg bw/day) was around 17-fold higher than that estimated for the workers in the first scenario. The systemic DNELs for the general and workers populations were 0.45 and 0.91 µg BPS/kg bw/day, respectively, which were 4.6- and 19-fold higher than the respective dermal DNELs. Risk Characterisation Ratio (RCR) (estimated exposure through urinary excretion compared with the systemic DNEL) in the first and most reliable scenario suggested that the risk was adequately controlled. In the second scenario, however, the RCR suggests that the risk might not be adequately controlled for both the general population and workers. This work raises the necessity of generate more toxicodynamic and toxicokinetic information, specially using dermal exposures, to properly assess the risk associated to dermal BPS exposure because the situation might presumably get worse after 2020.

Interactions of human butyrylcholinesterase with phenylvalerate and acetylthiocholine as substrates and inhibitors: kinetic and molecular modeling approaches.

Phenyl valerate (PV) is a substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Purified human butyrylcholinesterase (hBChE) showed PVase activity with a similar sensitivity to inhibitors as its cholinesterase (ChE) activity. Further kinetic and theoretical molecular simulation studies were performed. The kinetics did not fit classic competition models among substrates. Partially mixed inhibition was the best-fitting model to acetylthiocholine (AtCh) interacting with PVase activity. ChE activity showed substrate activation, and non-competitive inhibition was the best-fitting model to PV interacting with the non-activated enzyme and partial non-competitive inhibition was the best fitted model for PV interacting with the activated enzyme by excess of AtCh. The kinetic results suggest that other sites could be involved in those activities. From the theoretical docking analysis, we deduced other more favorable sites for binding PV related with Asn289 residue, situated far from the catalytic site ("PV-site"). Both substrates acethylcholine (ACh) and PV presented similar docking values in both the PV-site and catalytic site pockets, which explained some of the observed substrate interactions. Molecular dynamic simulations based on the theoretical structure of crystallized hBChE were performed. Molecular modeling studies suggested that PV has a higher potential for non-competitive inhibition, being also able to inhibit the hydrolysis of ACh through interactions with the PV-site. Further theoretical studies also suggested that PV could yet be able to promote competitive inhibition. We concluded that the kinetic and theoretical studies did not fit the simple classic competition among substrates, but were compatible with the interaction with two different binding sites.

Copper-dependent hydrolysis of trichloronate by turkey serum studied with use of new analytical procedure based on application of chiral chromatography and UV/Vis spectrophotometry.

Trichloronate is a racemic organophosphate, which has been used for the manufacture of insecticides. This compound induces delayed neuropathy in hen and humans. This study shows the Cu2+-dependent hydrolysis of trichloronate by turkey serum using UV/Vis spectrophotometry and chiral chromatography. The CHIRALCEL OD column and mobile phase of heptane allowed a resolution of 1.15 of its two enantiomers, while the liquid-liquid extraction showed a recovery of 95-98%. The optimum linear response was of 50 to 800 µM with a detection and quantification limit of 0.6 and 2 µM for (+)-trichloronate, and 0.7 and 2.3 µM for (-)-trichloronate. The levels of Cu2+-dependent hydrolysis (µM remaining concentration) quantified for 60 min at 37 °C and pH 7.4 were statistically higher (p ˂ 0.05) for (-)-trichloronate (65%) than (+)-trichloronate (32%). This stereoselective hydrolysis was confirmed by UV/Vis spectrophotometry using 2,4,5­trichlorophenol as standard, each of the enantiomers (93-95% purity) collected by HPLC, as well as aminoantipyrine and ferricyanide reagents to yield a colored product. This method exhibited an optimal linearity (r > 0.99) and a higher Cu2+-dependent hydrolysis (p < 0.05) to (-)-trichloronate (47%) than its corresponding (+)-form (31%). This results shows the Cu2+-dependent stereoselective hydrolysis of a racemic OP in its thio form (P = S) by an A-esterase of the turkey serum through the development of a colorimetric method and optimization of an existing chiral chromatographic method.

Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain.

Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In previous studies, in membrane chicken brain fractions, four components (EPα, EPß, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα is highly sensitive to mipafox and paraoxon, but is resistant to PMSF, and is spontaneously reactivated when inhibited with paraoxon. In this papers we shows that cholinesterase activities showed inhibition kinetic by PV, which does not fit with a competitive inhibition model when tested for the same experimental conditions used to discriminate the PVase components. Four enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in cholinesterase activity in the membrane fraction according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA. Components CP1 and CP2 could be related to EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.

Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and mipafox in human neuroblastoma SH-SY5Y cells.

The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.

Albumin, the responsible protein of the Cu2+-dependent hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) by chicken serum "antagonistic stereoselectivity".

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral analogous compound of the methamidophos insecticide that induces delayed neuropathy, and the R-(+)-HDCP enantiomer is an inhibitor of neuropathy target esterase (NTE). This enantiomer is not hydrolized by Ca2+-dependent phosphotriesterases in mammal tissues. Our group had reported R-(+)-HDCP hydrolysis in chicken serum enhanced by 30-250 µM copper in ex vivo assays, which we call "antagonistic stereoselectivity". We checked the hypothesis of the role of cupper binding proteins. Two hundred micrograms of human serum ceruloplasmine or horse kidney methallotionein in 1 mL containing 400 µM HDCP for 60 min showed no significant Cu2+-dependent hydrolysis. However under the same conditions, 10 µL of chicken serum or 10 µL of buffer containing 216 µg of chicken serum albumin (CSA) (amount of albumin content in this serum volume) with 100 µM Cu2+ showed the same stereoselectivity and similar levels to the Cu2+-dependent R-(+)-HDCP hydrolysis. About 75% of R-(+)-HDCP were hydrolyzed after 120 min in the presence of 100 µM Cu2+ (inhibited by 5 mM EDTA). No effects was observed by divalent cations Cu2+, Zn2+, Fe2+, Ca2+, Mn2+ and Mg2+. These results confirm that albumin is the protein responsible for "antagonistic stereoselectivity" observed in chicken serum.

Analysis of the neurotoxic effects of neuropathic organophosphorus compounds in adult zebrafish.

Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN.

Copper activation of organophosporus compounds detoxication by chicken serum.

Avian species contain low levels of enzymes that hydrolyze organophosphorus compounds (OPs), and chickens are used as a model of OPs delayed neurotoxicity. For both reasons, we studied the ability of chicken tissue for OP detoxication. A significant activating effect of Cu2+ on the hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was observed in hen plasma and the microsomal fractions of the liver, brain, and mainly in hen serum, by spectrophotometric and chiral chromatography methods. The concentration of 1 mM of Cu2+ or Zn2+ showed 200% and 168% activation, respectively, in hen plasma compared with the Ca2+-dependent hydrolysis, whereas these cations had an inhibitory effect on soluble liver and brain fractions. An increase of 1.5 to 19.5 fold in HDCP hydrolyzing activity was obtained for the 30-250 µM Cu2+ range when using chicken serum instead of hen plasma. This Cu2+-dependent hydrolysis in chicken serum was stereoselective for the R-(+)-HDCP isomer, which proved the opposite to the Ca2+-dependent stereoselective hydrolysis of the S-(-)-HDCP isomer reported in rat and rabbit serum. The level of copper needed to exert this effect should be further evaluated for its suitability for potential therapeutic and biotechnological applications.