Iris Mangas

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.

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.

Neurotoxic Effects Associated with Current Uses of Organophosphorus Compounds

Organophosphorus compounds (OPs) are a large and diverse class of chemicals that have been synthesized, since the XIX century for several purposes like chemical weapons, flame-retardants, ectoparasiticides and investigational new drugs, but mainly as agrochemicals in agriculture and indoor. Although the amount of OP pesticides being used is declining, especially in developed countries, OPs continue being one of the most important classes of insecticides and chemical warfare agents today due to its toxic effects on the enzyme acetylcholinesterase (AChE). Existing research on the toxicological effects of OPs is extensive, however, there is a lack of knowledge on the long-term effects of low levels of OPs and their exactly pathways of toxicity. Recent data prove that other molecular targets than AChE could be targeted by OPs, triggering these effects. Here these data are reviewed and it is highlighted that the current uses of OPs are producing several neurotoxic effects. It is also shown that, to protect people from possible uses and misuses of OPs, more regulations on OPs are needed. Moreover, more mechanistic studies are needed to completely understand their toxicological interactions and mechanisms of action and to identify the whole group of enzymes that interact with them.

Acetylcholine-hydrolyzing activities in soluble brain fraction: Characterization with reversible and irreversible inhibitors

Some effects of organophosphorus compounds (OPs) esters cannot be explained through actions on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In soluble chicken brain fraction, three components (Eα, Eβ and Eγ) of pheny lvalerate esterase activity (PVase) were kinetically discriminated and their relationship with acetylcholine-hydrolyzing activity (cholinesterase activity) were studied in previous works. In this work, four enzymatic components (CS1, CS2, CS3 and CS4) of cholinesterase activity have been discriminated in soluble fraction, according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA and to reversible inhibitors ethopropazine and BW284C51. Cholinesterase component CS1 can be related to the Eα component of PVase activity and identified as butyrylcholinesterase (BuChE). No association and similarities can be stablished among the other PVase component (Eβ and Eγ) with the other cholinesterase components (CS2, CS3, CS4). The kinetic analysis has allowed us to stablish a method for discriminating the enzymatic component based on a simple test with two inhibitors. It can be used as biomarker in toxicological studies and for monitoring these cholinesterase components during isolation and molecular identification processes, which will allow OP toxicity to be understood by a multi-target approach.

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, EPβ, 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.