Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phenyl valerate is used for detecting and measuring neuropathy target esterase (NTE) and has been used for discriminating esterases as potential target in hen model of organophosphorus delayed neuropathy. In previous studies we observed that phenyl valerate esterase (PVase) activity of an enzymatic fraction in chicken brain might be due to a butyrylcholinesterase protein (BuChE), and it was suggested that this enzymatic fraction could be related to the potentiation/promotion phenomenon of the organophosphate-induced delayed neuropathy (OPIDN). In this work, PVase activity of purified human butyrylcholinesterase (hBuChE) is demonstrated and confirms the novel observation that a relationship of BuChE with PVase activities is also relevant for humans, as is, therefore the potential role in toxicity for humans. The K and catalytic constant (k) were estimated as 0.52/0.72 µM and 45,900/49,200 min respectively. Furthermore, this work studies the inhibition by preincubation of PVase and cholinesterase activities of hBuChE with irreversible inhibitors (mipafox, iso-OMPA or PMSF), showing that these inhibitors interact similarly in both activities with similar second-order inhibition constants. Acethylthiocholine and phenyl valerate partly inhibit PVase and cholinesterase activities, respectively. All these observations suggest that both activities occur in the same active center. The interaction with a reversible inhibitor (ethopropazine) showed that the cholinesterase activity was more sensitive than the PVase activity, showing that the sensitivity for this reversible inhibitor is affected by the nature of the substrate. The present work definitively establishes the capacity of BuChE to hydrolyze the carboxylester phenyl valerate using a purified enzyme (hBuChE). Therefore, BuChE should be considered in the research of organophosphorus targets of toxicity related with PVase proteins. [ABSTRACT FROM AUTHOR]

Multiple epidemiological and experimental studies have demonstrated that exposure to organophosphorus compounds (OPs) is associated with a variety of neurological disorders. Some of these exposure symptoms cannot be precisely correlated with known molecular targets and mechanisms of toxicity. Most of the known molecular targets of OPs fall in the protein family of serine esterases. We have shown that three esterase components in the soluble fraction of chicken brain (an animal model frequently used in OP neurotoxicity assays) can be kinetically distinguished using paraoxon, mipafox and phenylmethyl sulfonyl fluoride as inhibitors, and phenyl valerate as a substrate; we termed them Eα, Eβ and Eγ. The Eα-component, which is highly sensitive to paraoxon and mipafox and resistant to PMSF, has shown sensitivity to the substrate acetylthiocholine, and to ethopropazine and iso-OMPA (specific inhibitors of butyrylcholinesterase; BChE) but not to BW 284C51 (a specific inhibitor of acetylcholinesterase; AChE). In this work, we employed a large-scale proteomic analysis B with a LC/MS/MS TripleTOF system; 259 proteins were identified in a chromatographic fractionated sample enriched in Eα activity of the chicken brain soluble fraction. Bioinformatics analysis revealed that BChE is the only candidate protein identified to be responsible for almost all the Eα activity. This study demonstrates the potential information to be gained from combining kinetic dissection with large-scale proteomics and bioinformatics analyses for identification of proteins that are targets of OP toxicity and may be involved in detoxification of phosphoryl and carbonyl esters. [ABSTRACT FROM AUTHOR]

Aluminium (Al), nickel (Ni), cadmium (Cd) and lead (Pb) levels in a total of 263 samples of 12 types of candies widely consumed in Spain, were evaluated. Samples were analysed using an ICP-MS method after acidic sample mineralization. Concentration ranges of Al, Ni, Cd and Pb were 21.28–62.91 µg g−1, 0.40–1.27 µg g−1, 0.12–1.01 µg g−1and 1.03–7.14 µg g−1, respectifgvely. Statistically significant positive correlations were calculated between concentrations of Ni–Al and Pb–Cd (p-values < 0.05). Taking into consideration the relatively high metal content, together with the high caloric density of these products, as well as high content of particular nutrients such as sugars that can induce development of certain pathologies like obesity and caries, indicate that frequent consumption of candy products is not recommended. [ABSTRACT FROM PUBLISHER]

O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) is a racemic organophosphate compound (OP) that induces delayed neuropathy in vivo. The O-hexyl 2,5-dichlorophenyl phosphoramidate R (R-HDCP) isomer inhibits and ages neuropathic target esterase (NTE) in hen brain. Moreover, human serum paraoxonase-1 (PON1) is a Ca-dependent enzyme capable of hydrolyzing OPs. The enzymatic activity of PON1 against OPs depends on the genetic polymorphisms present at position 192 (glutamine or arginine). The catalytic efficiency of PON1 is an important factor that determines neurotoxic susceptibility to some OPs. In the present study, we characterized the stereospecific hydrolysis of HDCP by alloforms PON1 Q192R human serum by chiral chromatography. Forty-seven human samples were characterized for the PON1 192 polymorphism. The hydrolysis data demonstrate that the three alloforms of PON1 show an exclusive and significant stereospecific Ca-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate S isomer (S-HDCP) at 19-127 µM at the concentrations that remain in all the samples. This stereoselective Ca-dependent hydrolysis of S-HDCP is inhibited by EDTA and is independent of the PON1 Q192R alloform. The present research reinforces the hypothesis that R-HDCP (an isomer that inhibits and causes NTE aging) is the enantiomer that induces delayed neuropathy by this chiral phosphoramidate due to the low hydrolysis level of the R-HDCP observed in this study. [ABSTRACT FROM AUTHOR]

Neuropathy target esterase (NTE) is involved in several disorders in adult organisms and embryos. A relationship between NTE and nervous system integrity and maintenance in adult systems has been suggested. NTE-related motor neuron disease is associated with the expression of a mutant form of NTE and the inhibition and further modification of NTE by organophosphorus compounds is the trigger of a delayed neurodegenerative neuropathy. Homozygotic NTE knockout mice embryos are not viable, while heterozygotic NTE knockout mice embryos yields mice with neurological disorders, which suggest that this protein plays a critical role in embryonic development. The present study used D3 mouse embryonic stem cells with the aim of gaining mechanistic insights on the role of Pnpla6 (NTE gene encoding) in the developmental process. D3 cells were silenced by lipofectamine transfection with a specific interference RNA for Pnpla6. Silencing Pnpla6 in D3 monolayer cultures reduced NTE enzymatic activity to 50% 20 h post-treatment, while the maximum loss of Pnpla6 expression reached 80% 48 h postsilencing. Pnpla6 was silenced in embryoid bodies and 545 genes were differentially expressed regarding the control 96 h after silencing, which revealed alterations in multiple genetic pathways, such as cell motion and cell migration, vesicle regulation, and cell adhesion. These findings also allow considering that these altered pathways would impair the formation of respiratory, neural, and vascular tubes causing the deficiencies observed in the in vivo development of nervous and vascular systems. Our findings, therefore, support the previous observations made in vivo concerning lack of viability of mice embryos not expressing NTE and help to understand the biology of several neurological and developmental disorders in which NTE is involved. [ABSTRACT FROM AUTHOR]

Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection, or potentiation/'promotion,' of organophosphorus delayed neuropathy (OPIDN), depending on whether it is dosed before or after an inducer of delayed neuropathy, such as mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and 'ongoing inhibition' (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I (20 min) of 35 and 198 μM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min). Four esterase components were globally identified considering also previously data with paraoxon and mipafox: EPα (4-8 %), highly sensitive to paraoxon and mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPβ (38-41 %), sensitive to paraoxon and PMSF, but practically resistant to mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39-48 %), paraoxon-resistant and sensitive to the micromolar concentration of mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed. [ABSTRACT FROM AUTHOR]

Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 μM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 μM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 μM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration. [ABSTRACT FROM AUTHOR]

This study measured inhalation exposure to 13 volatile organic compounds ( VOCs) among workers in the leatherwear industry in Spain, examined the changes in those exposures over a 5-year period, and documented local exhaust ventilation practices that affected exposure. In collaboration with an occupational risk prevention company, air samples were collected from 849 workers' personal breathing zones using personal air pumps with activated charcoal tubes. VOCs were analyzed using a GC/MS-optimized method modified in our laboratory from that proposed by Spanish authorities (INSHT). Airborne concentrations were compared with occupational exposure limit (OEL) values from the European authorities. The most frequently detected VOCs were acetone (98.1%), toluene (94.8%), n-hexane (71.2%) and other C6-C7 branched alkyl hydrocarbons (97.5%). Other frequently detected VOCs were MEK (64.9%), ethylacetate (60.7%), and cyclohexane (29.3%). Benzene was detected in 24.6% of samples. Although all the samples were taken while workers performed tasks judged to have the highest VOC exposure potential, only 14% of samples showed excessive aggregate exposure, and chemical-specific OELs were exceeded in a relatively small number of cases: 7.2% for n-hexane, 2.8% for toluene, 0.6% for acetone, and 0.4% for hexane isomers. Over the study period, a diminished use of n-hexane in solvent formulations and an increased use of branched hexane and heptane isomers were observed. Six factors relating to work location conditions and types were evaluated. Most high-exposure cases were associated with three task types. The presence of local exhaust ventilation was an important exposure control, but significant exposures despite the use of local exhaust were observed. Although n-hexane exposures significantly decreased over the study period, the overall level of VOC exposure did not decrease. More effective exposure prevention measures need to be implemented. [Supplementary materials are available for this article. Go to the publisher's online edition of Journal of Occupational and Environmental Hygiene for the following free supplemental resource: a file containing a descriptive analysis of the results, the influence of several workplace factors in the exposure and the accomplishment of the regulations in terms of occupational exposure limits.] [ABSTRACT FROM AUTHOR]

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus carbamates and sulfonyl fluorides) is sometimes unable to yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work, kinetic data were obtained for phenylmethylsulfonyl fluoride (PMSF) tested at different concentrations incubated for up to 3 h with soluble fraction of chicken peripheral nerve. PMSF is a protease and esterase inhibitor causing protection or potentiation of the organophosphorus-induced delayed neuropathy and is unstable in water solution. The target of the promotion effect was proposed to be a soluble esterase not yet identified. A kinetic model equation was deduced assuming a multienzymatic system with three different molecular phenomena occurring simultaneously: (1) inhibition, (2) spontaneous chemical hydrolysis of the inhibitor and (3) ongoing inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied for analyzing the experimental data. The best-fitting model is compatible with a resistant component (16.5-18%) and two sensitive enzymatic entities (both 41%). The corresponding second-order rate constants of inhibition ( ki = 12.04 × 10 and 0.54 × 10 μM min, respectively) and the chemical hydrolysis constant of PMSF ( kh = 0.0919 min) were simultaneously estimated. These parameters were similar to those deduced in fixed-time inhibition experiments. The consistency of results in both experiments was considered an internal validation of the methodology. The results were also consistent with a significant ongoing inhibition. The proportion of enzymatic components showed in this work is similar to those previously observed in inhibition experiments with mipafox, S9B and paraoxon, demonstrating that this kinetic approach gives consistent results in complex enzymatic systems. [ABSTRACT FROM AUTHOR]

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity. [ABSTRACT FROM AUTHOR]

Neuropathy Target Esterase (NTE) was initially identified as the primary target esterase of some organophosphorus compounds that cause delayed neuropathy. Some studies in vivo suggest that this protein may also perform a function in embryonic development and therefore also in cell differentiation. The aim of this work was to characterize embryonic stem cells (ESC) as cellular model before to approach to the role of NTE in embryotoxicity processes through mechanistic studies. Mouse D3 ESC in monolayer expressed an NTE activity of 23 nmol phenol/min/mg of protein, while mouse R1 ESC showed a specific NTE activity 3 times higher than D3. An increased expression of gene Pnpla6 (that codifies for NTE) was seen during differentiation in both the D3 cells in monolayer and embryonic bodies (EBS). The maximums of the Pnpla6 expression were reached after 30 h and 5 days of differentiation in monolayer and EBS cultures, respectively. This peak of the Pnpla6 expression correlated with the peak of the NTE enzymatic activity in D3 monolayers. NTE activity and Pnpla6 expression returned to basal levels after 48 h (in monolayer cultures) and 10 days (in EBS) of differentiation, respectively. The changes in the Pnpla6 expression did not correlate with changes noted in the expression of two endoderm, two ectoderm and one neuroectoderm gene markers. In conclusion, this manuscript reports about NTE expression in ESC and its variation during first stages of differentiation. Nevertheless, the role of this activity and the meaning of the variations detected during differentiation must be further studied. [ABSTRACT FROM AUTHOR]

Type B carboxylesterases (acetylcholinesterases, neuropathy target esterase, serine peptidases), catalyse the hydrolysis of carboxyl-ester substrates by formation of a covalent acyl-enzyme intermediate and subsequent cleavage and release of the acyl group. Organophosphorus compounds, carbamates, and others exert their mechanism of neurotoxicity by permanent covalent organophosphorylation or carbamylation at the catalytic site of carboxylesterases. Classical kinetic studies converted the exponential kinetic equation to a logarithmic equation for graphic analysis. This process, however, does not allow analysing complex situations. In this paper, kinetic model equations are reviewed and strategies developed for the following cases: (a) single enzyme, with classical linear equation; (b) multi-enzymatic system—discriminating several inhibitor-sensitive and inhibitor-resistant components; (c) ‘ongoing inhibition’—high sensitive enzymes can be significantly inhibited during the substrate reaction time, the model equations need a correction; (d) spontaneous reactivation (de-phosphorylation)—one or several components can be simultaneously inhibited and spontaneously reactivated; (e) spontaneous reactivation from starting time with the enzyme being partly or totally inhibited; (f) aging—single enzyme can be inhibited, spontaneously reactivated and dealkylating reaction (‘aging’) simultaneously occurs; and (g) aging and spontaneous reactivation from starting time with the enzyme being partly or totally inhibited. Analysis of data using the suggested equations allows the deduction of inhibition kinetic constants and the proportions of each of the enzymatic components. Strategies for practical application of the models and for obtaining consistent kinetic parameters, using multi-steps approaches or 3D fitting, are presented. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Ca2+-dependent and EDTA-resistant hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and paraoxon was studied in serum and subcellular fractions of liver, kidney and brain of hen, rat and rabbit. HDCP was the best substrate among all the tissues studied, except that of rabbit serum which showed the highest Ca2+-dependent paraoxon hydrolysing activity (paraoxonase). High HDCP hydrolysing activity (HDCPase) was detected in the brain tissue of the three species studied, whereas low or no paraoxonase was found. The HDCPase/paraoxonase ratio of Ca2+-dependent hydrolysing activities ranged from 0.5 to 83 for tissues of the same species. EDTA-resistant HDCPase activity was more than 50% of the total activities in hen tissues, with an almost undetectable Ca2+-dependent paraoxonase activity in most organs. The same response was observed in rat tissues, except for serum where the Ca2+-dependent HDCPase and paraoxonase activities were higher (70 and 25% of total activities, respectively). EDTA-resistant HDCPase and paraoxonase activities represented less than 25% of all activities in rabbit tissues. Paraoxon has traditionally been the substrate for measuring organophosphorus hydrolysing activities. However, HDCP could be a good substrate in addtion to paraoxon for monitoring other phosphotriesterases in biological tissues. [ABSTRACT FROM AUTHOR]

The hydrolysis of carbaryl by bovine serum albumin (BSA) was studied at toxicologically relevant concentrations (range 15–300 μM) in order to determine the role of this protein in the detoxication of the carbamate in vivo. The 1-naphthol released during the hydrolysis of carbaryl was monitored using gas chromatography coupled with mass spectrometry. BSA hydrolyzed carbaryl in a time-progressive way. The hydrolysis was also dependent of enzyme (1.0, 2.5, 5.0 and 7.0 mg ml−1) and substrate (range between 15 and 1,000 μM) concentration. The estimated turnover number and Michaelis–Menten constant were 1.6 × 10−4 s−1 and 430 μM, respectively. Thus, the second order rate constant was 0.37 M−1 s−1. At enzyme concentrations of 7.0 mg ml−1 and substrate concentrations ranging between 50 and 300 μM about 80% of substrate was hydrolyzed in 3 h. At lower substrate concentrations (15 and 30 μM carbaryl) also significant hydrolysis was detected at the highest enzyme concentration, even when these substrate concentrations were 30 and 15 times lower than the Michaelis–Menten constant. Although the efficacy of the enzymatic hydrolysis is low, the extrapolation of our results to the physiological albumin high concentrations (around 40 mg ml−1) suggests that the hydrolysis of carbaryl by serum albumins plays a critical role in the detoxication of this carbamate at in vivo toxicologically relevant concentrations. [ABSTRACT FROM AUTHOR]

Human serum (HS) and human serum albumin (HSA) were able to hydrolyse the carbamate carbaryl. Carbarylase activity found in I-ISA was slightly activated by 1 mM Zn2+, Mn2+, Cd2+, Ni2+ and Na+ and by 0.01 mM Pb2+. The organophosphorus compounds paraoxon and O-hexyl O-2,5-dichlorophenyl phosphoramidate, caprylic acid, palmitic acid and the carboxyl ester p-nitrophenyl butyrate inhibited the hydrolysis of carbaryl by HSA, being in the last case a competitive inhibition. Using selective amino acid reagents, we concluded that Cys, Trp, Arg and Tyr seem to play important roles in the carbarylase activity of HSA. In addition, Tyr and Arg seem to be located in the active centre of the enzyme since carbaryl protected the activity from the inhibition. It was concluded that HSA hydrolyses carbaryl by a mechanism similar to that described for rabbit serum albumin based in transient carbamylation of a Tyr residue. The extrapolation of the hydrolysis rate to physiological albumin concentrations suggests that albumin might be playing a critical role in the detoxication of carbaryl. [ABSTRACT FROM AUTHOR]

The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 ± 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate ( p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates. [ABSTRACT FROM AUTHOR]

Hens were given a single oral dose (0.235 mg/kg) of tri- o-cresylphosphate (TOCP) during chronic n-hexane treatment (200 mg/kg daily, 5 days/week). They were compared with other animals treated only with n-hexane or only with TOCP. Animals treated with a higher TOCP dose (1 ml/kg) were used as positive controls. The animals treated with both n-hexane and TOCP showed rapid development of severe ataxia. The rate of the ataxia development was similar to that of the positive controls but with earlier onset of the first signs and with less loss of body weight. However, animals treated only with n-hexane, under the same conditions, showed only reversible weakness and sedative effects, and those treated only with TOCP showed slow and slight ataxia development. The n-hexaneand TOCP-treated hens showed axonal swelling with myelin retraction associated with Ranvier's nodes, which is characteristic of long hexacarbon exposure. Some internodal swellings were also observed but less frequently than the paranodal swellings. The time course of the ataxia development was similar to an organophosphorus-induced delayed neuropathy (OPIDN); however, the light microscopy observation more closely resembled hexacarbon neuropathy. The results suggest a potentiation effect of n-hexane and TOCP neurotoxicities which could be related to some human occupational neuropathies. [ABSTRACT FROM AUTHOR]

Cholinesterase (ChE) inhibitors have been described in the distillation residue of hexane and other industrial solvents. The residue of a commercial hexane has been fractionated by preparative chromatography. The anticholinesterase (antiChE) activity was isolated in two fractions (F-5, F-7) which contained only 0.61 and 0.16% respectively of the original dry weight hexane residue. In the former fraction, reversible and irreversible progressive inhibition was observed, and organophosphorus compounds (OPs) were detected colorimetrically and by gas chromatography. This fraction was subfractionated in a second chromatographic step. One subfraction containing the highest antiChE activity and 88% phosphorus of F-5 was isolated. In this subfraction triphenylphosphate and other not definitely identified OP compounds were detected by gas chromatography/mass spectrometry, together with several adipates and phthalates, including di- n-butylphthalate. This phthalate could explain the reversible inhibition of ChE by the hexane residue, and triphenylphosphate and the unidentified OP the irreversible inhibition. A possible toxicological role of these impurities is discussed in relation to occupational neuropathies by exposure to solvents. [ABSTRACT FROM AUTHOR]

Commercial hexane was concentrated by distillation. The distillation residue (0.43 ml/l original solvent) contains material which inhibits human serum cholinesterase (ChE) 'in vitro' with a slight effect on acetylcholinesterase. Phosphorus was detected equivalent to 0.33 μmol monophosphorus compound/litre original solvent. The inhibition was progressive with the enzyme-inhibitor preincubation time. A partial reactivation of the inhibited enzyme was obtained by treatment with hydroxylamine and 2-PAM. The results are coherent with a covalent inactivation by more than one inhibitor which acylate (probably phosphorylate) ChE, although it seems likely that a reversible but unstable inhibitor could also be present in the hexane residue. The results are discussed in the context of the known neurotoxic effects of n-hexane and some organophosphorus esters. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chiral analogous compound of methamidophos insecticide are only poorly hydrolyzed by Ca2+-dependent phosphotriesterases in mammals tissues including the human serum. We reported the hydrolysis of O-hexyl O-2,5- dichlorophenyl phosphoramidate (HDCP) in chicken serum. The hydrolysis of the R-(+)-HDCP isomer is strongly increased in vitro in the presence of 30-250 µM copper. It is the opposite estereoselectivity of that showed by liver Ca2+-dependent activity. We name it as "antagonistic stereoselectivity". Diluted chicken serum (10 µL in 1 mL solution of 400 µM HDCP) or the equivalent amount of commercial chicken serum albumin (CSA 216 µg/mL) with 100 µM Cu2+, showed about 50% and 75% of R-(+)-HDCP hydrolysis after 60 and 120 min. In the same conditions other commercial serum metalloproteins with high affinity to Cu2+ (cuproproteins) as human serum ceruloplasmin or horse kidney metallothionein did not showed significant Cu2+-dependent hydrolysis. Moreover, other divalent cations (Zn2+, Fe2+, Ca2+, Mn2+ and Mg2+) did not showed this activation. The results confirm that the CSA is the protein responsible of "antagonistic stereoselectivity" that had been observed in the chicken serum. The effect of copper on the hydrolysis of HDCP by other animal albumins is shown in this work. [ABSTRACT FROM AUTHOR]

Protective mechanism against organophosphorus compounds (OPs) toxicity are mainly based in molecular processes frequently divided conceptually in (A) catalytic and (B) non-catalytic bio-scavengers. Modified natural proteins and small molecules have been developed for applying in therapy and protection. The catalytic ones are mainly associate to the classical concept of A-esterases (phosphotriesterases, PTEs, i.e. paraoxonase); they hydrolyze carboxylesters and OPs by a divalent cation dependent mechanism. The non-catalytic scavengers are mainly associated to covalent binding to proteins, especially B-esterases with a serine or tyrosine residue, which hydrolyzes carboxylesters. However, if an OP is bound (organophosphorylation), its represents an enzymatic inhibition in some cases considered "the target" of toxicity or initial molecular event (IME) in their mode of action developing toxicity (adverse output pathway, AOP). The binding to proteins also represents a sequestration avoiding the OP interaction to other protein. However, there are protein binding OPs (non-catalytic bioscavengers) which can be slowly dephosphorylated, having a role as catalytic scavenger. A proportion of B-esterase activity in serum and brain shows reversible inhibition and their protective role just in situ in the target tissue of toxicity need to be investigated. Serum albumin is other example of B-esterase mainly thorough a tyrosine residue; its role in detoxication have been demonstrated and adducts applied as biomarker of exposure. Moreover, for a specific phosphoramidate family hydrolysis capacity may be enhanced by copper, probably by a mechanism not related with its B-esterase activity. Therefore, we have examples in the borderline between non-catalytic and catalytic scavengers. [ABSTRACT FROM AUTHOR]

The toxicity of organophosphorus compounds (OPs) cannot be explained only by action on acetylcholinesterase or neuropathy target esterase (NTE). A fraction of the membrane bound phenylvalerate esterase activity (PVase) is associated to NTE, the key initiating molecular event in the OP-induced delayed neuropathy (OPIDN). An enzymatic fraction in chicken brain soluble PVase has been reported to be due to a butyrylcholinesterase protein, and we suggested that this enzymatic fraction could be related to the mode of action of the potentiation/promotion phenomenon of the OPIDN. We showed that human butyrylcholinesterase (hBuChE) shows PVase activity. Mipafox, iso-OMPA or PMSF inhibited both activities with similar kinetic constants for both activities. Moreover, the substrates acethylthiocholine and phenyl valerate showed competition in their activities. The results suggest that both activities are related to the same active center. This work studies in depth the kinetic interactions between phenyl valerate and acetylthiocholine in human butyrylcholinesterase, showing that the interactions are different to the competitive model of substrates according to the Michaelis-Menten reaction. The approach introduced in this work suggests that other site could be involved in the interaction with phenyl valerate. In addition, we have observed that human acetylcholinesterase has also phenyl valerate esterase activity, but with lower activity than human butyrylcholinesterase. The level of phenylvalerate esterase activity in cholinesterases depends on the species and the type of cholinesterase. Further evaluation of the molecular interactions is under study. [ABSTRACT FROM AUTHOR]

The enzymes that hydrolyze organophosphorus compounds are called phosphotriesterases. The presence of phosphotriesterases has been described in a variety of tissues. The physiological role of these enzymes is not known, although a clear correlation exists between the levels of phosphotniesterases and susceptibility of the species to the toxiceffects of organophosphorus compounds. Thus, mammals that high levels of phosphotriesterases in serum and liver are more tolerant to the toxic effects of these compounds than birds and insects -- these being species considered lacking of phosphotriesterases. Because most of these enzymes are not well characterized, they are usually differentiated according to their different patterns of response to activators and/or inhibitors. Phosphotriesterases usually depend of divalent cations and therefore EDTA usually inhibits them. A peculiar EDTA-resistant phosphotriesterase has been described in serum albumin. The biotechnological and therapeutical applications of phosphotriesterases arecurrently subject to study. [ABSTRACT FROM AUTHOR]