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.

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.

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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.

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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 × 10⁻² and 0.54 × 10⁻² μM⁻¹ min⁻¹, respectively) and the chemical hydrolysis constant of PMSF (kh = 0.0919 min⁻¹) 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.

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.