Jorge Estévez

Model equations for the kinetics of covalent irreversible enzyme inhibition and spontaneous reactivation: Esterases and organophosphorus compounds

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.

An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods

The main available alternatives for testing embryotoxicity are cellular tests with stem cells and in vitro-ex vivo tests with embryos. In cellular tests, the most developed alternative is the embryonic stem cell test, while the most developed tests involving embryos are the zebrafish and whole embryo culture test. They are technically more complex than cellular tests, but offer the advantage of determining the expectable phenotypic alteration caused by the exposure. Many efforts are currently being made, basically through proteomic and genomic approaches, in order to obtain improvements in predictivity of these tests. Development is a very complex process, and it is highly unlikely that a single alternative test can yield satisfactory performance with all types of chemicals. We propose a step-wise approach where model complexity, and consequently technical skills and economical costs, gradually increase if needed. The first level would be run short cellular assays to detect effects in early differentiation stages. The second level would involve longer cellular embryotoxicity tests to search embryotoxicants that have an effect on late differentiation stages. The third stage would consider tests with embryos because they allow the determination of hazards based on molecular and morphological alterations, and not only on differentiating cells.

Phenylmethylsulfonyl fluoride, a potentiator of neuropathy, alters the interaction of organophosphorus compounds with soluble brain esterases.

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. The molecular target of promotion has not yet been identified. Kinetic data of esterase inhibition were first obtained for PMSF with a soluble chicken brain fraction and then analyzed using a kinetic model with a multienzymatic system in which inhibition occurred with the simultaneous chemical hydrolysis of the inhibitor and ongoing inhibition (inhibition during the substrate reaction). The best fitting model was a model with resistant fraction, Eα (28%), and two sensitive enzymatic entities, Eβ (61%) and Eγ (11%), with I(50) at 20 min of 70 and 447 μM, respectively. The estimated constant of the chemical hydrolysis of PMSF was kh = 0.23 min(-1). Eα, which is sensitive to mipafox and resistant to PMSF, became less sensitive to mipafox when the preparation was preincubated with PMSF. Its Eα I(50) (30 min) of mipafox increased with the PMSF concentration used to preincubate it. Eγ is sensitive to both PMSF and mipafox, and after preincubation with PMSF, Eγ became less sensitive to mipafox and was totally resistant after preincubation with 10 μM PMSF or more. The sensitivity of Eα to paraoxon (I(50) 30 min from 9 to 11 nM) diminished after PMSF preincubation (I(50) 30 min 185 nM) and showed no spontaneous reactivation capacity. The nature of these interactions is unknown but might be due to covalent binding at sites other than the substrate catalytic center. Such interactions should be considered to interpret the potentiation/promotion phenomenon of PMSF and to understand the effects of multiple exposures to chemicals.

Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of serum esterases are very similar to those of soluble fraction of peripheral nerves. So, serum PVases could be considered as appropriate biomarkers, as a mirror for the neural soluble paraoxon and mipafox sensitive soluble esterases that could be used for biomonitoring purpose.

Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain.

Organophosphorus compounds (OPs) induce neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, OPs interact with other esterases of unknown biological function. In soluble chicken brain fractions, three components of enzymatic phenylvalerate esterase activity (PVase) called Eα, Eβ and Eγ, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Eα PVase activity (resistant PVase activity to 1500 μM PMSF for 30 min) was tested with different acetylthiocholine concentrations, inhibition was observed. The best-fitting model to the data was the non-competitive inhibition model (Km=0.12, 0.22 mM, Ki=6.6, 7.6 mM). Resistant acetylthiocholine-hydrolyzing activity to 1500 μM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11 mM; Ki=1.7, 2.2 mM). Eβ PVase activity (resistant PVase activity to 25 μM mipafox for 30 min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25 μM mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06 mM; Ki=0.44, 0.58 mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins.

New insights on molecular interactions of organophosphorus pesticides with esterases.

Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the organophosphorus toxicity.

Cholinesterase assay by an efficient fixed time endpoint method.

Highlights • An end-point method for cholinesterase determinations is performed.• This method is based on stopping the reaction after a fixed reaction time.• A large number of samples can be processed for complex kinetic assays.• This assay can also be applied for manual, or with, automated workstations.• This procedure allows to avoid undesired reactions by DTNB and TNB.

Butyrylcholinesterase identification in a phenylvalerate esterase-enriched fraction sensitive to low mipafox concentrations in chicken brain

Abstract 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.

Effects of mipafox, paraoxon, chlorpyrifos and its metabolite chlorpyrifos-oxon on the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells

Chlorpyrifos (CPS) is an organophosphorus compound (OP) capable of causing well-known cholinergic and delayed syndromes through the inhibition of acetylcholinesterase and Neuropathy Target Esterase (NTE), respectively. CPS is also able to induce neurodevelopmental toxicity in animals. NTE is codified by the Pnpla6 gene and plays a central role in differentiation and neurodifferentiation. We tested, in D3 mouse embryonic stem cells under differentiation, the effects of the NTE inhibition by the OPs mipafox, CPS and its main active metabolite chlorpyrifos-oxon (CPO) on the expression of genes Vegfa, Bcl2, Amot, Nes and Jun, previously reported to be under- or overexpressed after Pnpla6 silencing in this same cellular model. Mipafox did not significantly alter the expression of such genes at concentrations that significantly inhibited NTE. However, CPS and CPO at concentrations that caused NTE inhibition at similar levels to mipafox statistically and significantly altered the expression of most of these genes. Paraoxon (another OP with capability to inhibit esterases but not NTE) caused similar effects to CPS and CPO. These findings suggest that the molecular mechanism for the neurodevelopmental toxicity induced by CPS is not based on NTE inhibition, and that other unknown esterases might be potential targets of neurodevelopmental toxicity.