Detoxification Of Organophosphates Research Articles

Organophosphate Detoxification and Acetylcholinesterase Reactivation Triggered by Zeolitic Imidazolate Framework Structural Degradation.

Organophosphate (OP) toxicity is related to inhibition of acetylcholinesterase (AChE) activity, which plays a key role in the neurotransmission process. In this work, we report the ability of different zinc zeolitic imidazolate frameworks (ZIFs) to behave as potential antidotes against OP poisoning. The Zn-L coordination bond (L = purine, benzimidazole, imidazole, or 2-methylimidazole) is sensitive to the G-type nerve agent model compounds diisopropylfluorophosphate (DIFP) and diisopropylchlorophosphate, leading to P-X (X = F or Cl) bond breakdown into nontoxic diisopropylphosphate. P-X hydrolysis is accompanied by ZIF structural degradation (Zn-imidazolate bond hydrolysis), with the concomitant release of the imidazolate linkers and zinc ions representing up to 95% of ZIF particle dissolution. The delivered imidazolate nucleophilic attack on the OP@AChE adduct gives rise to the recovery of AChE enzymatic function. P-X bond breakdown, ZIF structural degradation, and AChE reactivation are dependent on imidazolate linker nucleophilicity, framework topology, and particle size. The best performance is obtained for 20 nm nanoparticles (NPs) of Zn(2-methylimidazolate)2 (sod ZIF-8) exhibiting a DIFP degradation half-life of 2.6 min and full recovery of AChE activity within 1 h. 20 nm sod ZIF-8 NPs are not neurotoxic, as proven by in vitro neuroblastoma cell culture viability tests.

Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates.

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 μM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD50 shift was 23.7xLD50 for prophylaxis and 8xLD50 for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

Strategies for enhanced bioavailability of oxime reactivators in the central nervous system.

Oxime reactivators of acetylcholinesterase are commonly used to treat highly toxic organophosphate poisoning. They are effective nucleophiles that can restore the catalytic activity of acetylcholinesterase; however, their main limitation is the difficulty in crossing the blood-brain barrier (BBB) because of their strongly hydrophilic nature. Various approaches to overcome this limitation and enhance the bioavailability of oxime reactivators in the CNS have been evaluated; these include structural modifications, conjugation with molecules that have transporters in the BBB, bypassing the BBB through intranasal delivery, and inhibition of BBB efflux transporters. A promising approach is the use of nanoparticles (NPs) as the delivery systems. Studies using mesoporous silica nanomaterials, poly (L-lysine)-graft-poly(ethylene oxide) NPs, metallic organic frameworks, poly(lactic-co-glycolic acid) NPs, human serum albumin NPs, liposomes, solid lipid NPs, and cucurbiturils, have shown promising results. Some NPs are considered as nanoreactors for organophosphate detoxification; these combine bioscavengers with encapsulated oximes. This study provides an overview and critical discussion of the strategies used to enhance the bioavailability of oxime reactivators in the central nervous system.

Efficiency of Several Cytochrome P450 Biomarkers in Highlighting the Exposure of Daphnia magna to an Organophosphate Pesticide.

The cytochromes P450 (CYP450) represent a major enzyme family operating mostly in the first step of xenobiotic detoxification in aquatic organisms. The ability to measure these CYP450 enzymes’ activities provides a crucial tool to understand organisms’ response to chemical stressors. However, research on CYP450 activity measurement is still limited and has had variable success. In the present study, we optimize, compile, and compare existing scientific information and techniques for a series of CYP450 biomarkers (EROD, MROD, ECOD, APND, and ERND) used on Daphnia magna. Additionally, we explored these CYP450 biomarkers’ activities through the first 5 days of life of daphnids, providing a link between their age and sensitivity to chemicals. In the experiment, daphnids were exposed to an organophosphate pesticide (diazinon) from birth to measure the molecular response of the detoxification process. Our results suggest EROD as the most applicable biomarker for organisms such as D. magna, with a higher organophosphate detoxification rate in daphnids that are 2 and 5 days old. Additionally, a larger body size allowed a more accurate EROD measurement; hence, we emphasize the use of 5-day-old daphnids when analyzing their detoxification response.

Insights of OPs and PYR cytotoxic potential Invitro and genotoxic impact on PON1 genetic variant among exposed workers in Pakistan

Different pesticide chemicals are used to enhance crop yield by protecting from pests. Organophosphate (OPs) and Pyrethroid (PYR) are used in fields of Sanghar, Sindh Pakistan. PON1 an antioxidant enzyme implicated in OPs detoxification may predispose by OPs chronic exposure. This study was conducted to evaluate the toxic potential of active pesticide chemicals at cellular and genetic levels. To examine toxic potential, locally consumed pesticide n = 2 and reference pesticide compounds organophosphate (OPs): Chloropyrifos, Malathion and Pyrethroid (PYR): Cyprmethrin, Cyhalothrin n = 4 were tested against NIH 3T3 cells using MTS assay. Local consumer pesticides demonstrated relevance for half-maximum inhibitory concentration (IC50) 0.00035 mg/mL with selected compound. Malathion IC50 exhibited the highest cytotoxicity among four compounds at 0.0005 mg/mL. On genotoxicity analysis in exposed subjects n = 100 genotypes and alleles n = 200 exhibited significant differences in genotypic and allelic frequencies of pesticide exposed subjects and controls n = 150 (X2 = 22.9, p = 0.001). Screening of genotypes were performed by PCR- RFLP. Statistical assessment carried out using online software and tools. Results suggested that higher heterozygous genotype A/G (74%) may confer low PON1 metabolic activity towards pesticides in exposed subjects. Findings could be helpful to establish health plans by avoiding toxic chemicals that harming exposed population.

Enzyme Nanoreactor for In Vivo Detoxification of Organophosphates.

A nanoreactor containing an evolved mutant of Saccharolobus solfataricus phosphotriesterase (L72C/Y97F/Y99F/W263V/I280T) as a catalytic bioscavenger was made for detoxification of organophosphates. This nanoreactor intended for treatment of organophosphate poisoning was studied against paraoxon (POX). Nanoreactors were low polydispersity polymersomes containing a high concentration of enzyme (20 μM). The polyethylene glycol-polypropylene sulfide membrane allowed for penetration of POX and exit of hydrolysis products. In vitro simulations under second order conditions showed that 1 μM enzyme inactivates 5 μM POX in less than 10 s. LD50-shift experiments of POX-challenged mice through intraperitoneal (i.p.) and subcutaneous (s.c.) injections showed that intravenous administration of nanoreactors (1.6 nmol enzyme) protected against 7 × LD50 i.p. in prophylaxis and 3.3 × LD50 i.p. in post-exposure treatment. For mice s.c.-challenged, LD50 shifts were more pronounced: 16.6 × LD50 in prophylaxis and 9.8 × LD50 in post-exposure treatment. Rotarod tests showed that transitory impaired neuromuscular functions of challenged mice were restored the day of experiments. No deterioration was observed in the following days and weeks. The high therapeutic index provided by prophylactic administration of enzyme nanoreactors suggests that no other drugs are needed for protection against acute POX toxicity. For post-exposure treatment, co-administration of classical drugs would certainly have beneficial effects against transient incapacitation.

Organophosphate detoxification by membrane-engineered red blood cells

Biotherapeutics have achieved global economic success due to their high specificity towards their drug targets, providing exceptional safety and efficiency. The ongoing shift away from small molecule drugs towards biotherapeutics heightens the need to further improve the pharmacokinetics of these biological drugs. Three pervasive obstacles that limit the therapeutic capacity of biotherapeutics are proteolytic degradation, circulating half-life, and the development of anti-drug antibodies. These challenges can culminate in limited efficiency and consequently warrant the need for higher drug doses and more frequent administration. We have explored the coupling of biotherapeutics to long-lived and biocompatible red blood cells (RBCs) to address limited pharmacokinetics. Butyrylcholinesterase (BChE), for example, provides prophylactic protection against organophosphate nerve agents (OPNAs), yet the short circulation life of the drug requires extraordinary doses. Herein, we report the rapid and tunable chemical engineering of BChE to RBC membranes to create a cell-based delivery system that retains the enzyme activity and enhances stability. In a three-step process that first pre-modifies BChE with a cell-reactive polymer chain, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to the surface of each RBC without diminishing the bioscavenging capacity of the enzyme. Critically, this membrane-engineering approach was cell-tolerated with minimal hemolysis observed. These results provide strong evidence for the ability of engineered RBCs to serve as an enhanced biotherapeutic delivery vehicle. Statement of SignificanceOrganophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme's activity and enhancing its stability.

FEATURES OF ORGANOPHOSPHATES IMMOBILIZATION VIA STREPTAVIDIN-BIOTIN SYSTEM FOR EXPERIMENTS ON SELECTION OF APTAMERS

Poisoning with organophosphorus compounds occupy one of the leading places in exotoxicosis. At the first stage, the detoxification of organophosphates can be provided with the help of DNA or RNA aptamers that bind the poison in the bloodstream. Currently, the main method of searching for aptamers is the experimental method of systematic evolution of ligands by exponential enrichment (SELEX). In the process of aptamer selection, the target molecule must be immobilized via the streptavidin-biotin complex. Since the poison molecule is small in size, to increase its availability for binding to aptamer, it is necessary to use a spacer between organophosphorus compounds and biotin. The aim of this work was to optimize the selection of aptamers for organophosphorus compounds by increasing the availability of a poison molecule immobilized via the streptavidin-biotin complex on the example of paraoxon. For this purpose, three spacers between organophosphorus compounds and biotin were tested using molecular modeling methods: three links of polyethylene glycol (3-PEG), four links of polyethylene glycol (4-PEG) and aminohexyl. The conformation of the biotinylated paraoxon complex with streptavidin and the interaction of paraoxon with the binding fragment of the aptamer were modeled using molecular docking and molecular dynamics methods. The ability of biotinylated paraoxon to bind to the aptamer has been evaluated by analyzing the surface area of the paraoxon available to the solvent, as well as by calculating the free binding energies. It has been shown that only in the case of aminohexyl immobilized paraoxon can contact the aptamer. At the final stage, the synthesis of paraoxon bound to biotin via aminohexyl was carried out.

Ultra-fast catalytic detoxification of organophosphates by nano-zeolitic imidazolate frameworks

Detrimental and injurious impacts of Organophosphates that have had on environment, humans, organisms and the other animals or plants have not been surreptitious to anyone worldwide. Nevertheless, up to now, among many efforts that have been devoted to detoxification of Organophosphates (OPs), catalytic detoxification has been the most applicable, cost-effective, efficacious and safest way to break down these dangerous materials. Herein, the utilization of zeolitic imidazolate frameworks (ZIFs), for the first time, has been reported to deactivate Diazinon as an organophosphate agent demonstrated at room temperature. In the following research, the catalysts were analyzed by PXRD, FT-IR, FE-SEM, BET, CO2 adsorption/desorption and TG. The decontamination processes were followed by 31P NMR, HPLC, and UV–vis to evaluate catalytic efficiency. Interestingly, supreme reusability, durability and potentially stunning catalytic activity represent them as alternate materials for their amazing elimination of OPs compared to the other MOFs.

The Effect of Resveratrol on Binding and Esterase Activity of Human and Rat Albumin

The directional effect on albumin of the molecules which modulate its binding and/or esterase activity could allow stoichiometric and/or catalytic detoxification of organophosphates in the bloodstream. In this study, the effect of polyphenols on binding and catalytic activity of human (HSA) and rat (RSA) serum albumins has been investigated by molecular modeling on the examples of trans-resveratrol and paraoxon. Molecular docking of the paraoxon molecule into the Sudlow II site of HSA and RSA was followed by a calculation of conformational changes and free energies of the albumin-paraoxon complex formation using molecular dynamics simulation. The same calculation was carried out in the presence of a trans-resveratrol molecule in the Sudlow I site. It has been shown that the effect of resveratrol only consists in decreasing pseudo-esterase activity of RSA towards paraoxon.

Targeted catalytic degradation of organophosphates: pursuing sensors

Abstract Chemical security has been a hot topic over several years, especially involving organophosphates (OP), which are related to organophosphorus chemical warfare and pesticides. The main challenges are to develop efficient ways to destroy stockpiles of these materials and also to monitor their presence in the field or food. A promising approach in this sense is the targeted design of catalysts that can react with OP and degrade them. Herein, we present a summary of some recent advances towards OP detoxification and monitoring that involves four different approaches: (i) How? Understanding the mechanism: thorough mechanistic elucidation and design of an ideal catalytic scaffold; (ii) Nanocatalysts and sensors: rational functionalization of carbon nanomaterials leading to nanocatalysts as powder and thin films. A surface-enhanced Raman scattering (SERS) sensor for OP was also obtained combining the functionalized material with silver nanoparticles; (iii) Catalysts from waste: reuse of rice husk waste as well as waste derived from the cheap gum arabic for developing biocatalysts that have high added-value and are environmentally friendly; (iv) A simple sensor: a simple, cheap and compact homemade colorimeter that can be used as a colorimetric sensor for detecting quantitatively various compounds, including pesticides.

Building a three-dimensional model of rat albumin molecule by homology modeling

Albumin is known as being able to cleave ester bonds in organophosphates (OP) and serve as a major means of OP detoxification due to its large amount in the blood serum. Most of in vivo toxicological studies are conducted on rodents, mice and rats. To adequately extrapolate results of ligand/albumin interaction studies to a human organism, it is necessary to carry out a comparative analysis of the rat (RSA) and human (HSA) albumin structure by in silico methods. X-ray diffraction analysis of RSA has not been done as yet, and the RSA structure is still undetermined. The aim of this study was to build a three-dimensional (3D) model of RSA by homology modeling. As templates, 14 RSA homologs were selected from the Protein Data Bank. A 3D model of the RSA molecule was built using the RSA primary sequence and 3D models of the selected templates. The 3D model geometry was improved by the energy minimization method. The quality of the model was evaluated by 9 parameters. According to this evaluation, in 97% of protein structures from the comparative dataset these parameters were worse than in the obtained model. It was concluded that this model is suitable for further research, specifically, for in silico studies of the RSA/OP interaction and comparative evolutionary analysis of RSA and HSA.

GENETIC POLYMORPHISMS OF GLUTATHIONE STRANSFERASE (M1 AND T1) AND PARAOXONASE 1 (PON1) AND SUSCEPTIBILITY TO CHRONIC KIDNEY DISEASE OF UNKNOWN ETIOLOGY IN PESTICIDE EXPOSED PATIENTS AT ZAGAZIG UNIVERSITY HOSPITALS

Background: Pesticide compounds are commonly used worldwide and in Egypt. Chronic low level exposure of humans to these compounds has resulted in many health adverse effects. There are many reports relating chronic kidney disease of unknown etiology (CKDu) epidemic to pesticide exposure including organophosphates (OP). Glutathione-S-transferase mu (GSTM1) and glutathione-S-transferase theta (GSTT1) are phase II xenobiotic metabolizing enzymes. Paraxonase 1 (PON1) is an enzyme linked to high-density lipoprotein. It can prevent oxidation of low-density lipoproteins and participates in organophosphate detoxification. Objective: To investigate the association of genetic polymorphism of GST (M1, T1) and PON1 Q192R with CKDu in pesticide exposed patients presented to Nephrology Unit, Internal Medicine Department, Zagazig University Hospitals, Sharkia Governorate. Methods: Gas chromatography- mass spectrometry (GC-MS) was used for detection of dialkyl phosphate metabolites in urine samples of subjects included in the study then genomic DNA isolated from the blood samples of 48 CKDu patient chronically exposed to pesticides giving positive result for dialkyl phosphate metabolites and matched control group was used to detect the presence or absence of the GSTM1 and GSTT1 genes (gene deletion) by multiplex polymerase chain reaction technique (multiplex PCR) and PON1 polymorphisms using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Conclusion: This study suggested that there is a correlation between chronic exposure to organophosphates and the risk of CKDu due to GSTM1 and GSTT1 genes deletions and PON1 gene polymorphisms.

Detoxification of organophosphates using imidazole-coated Ag, Au and AgAu nanoparticles

Organophosphate (OP) detoxification is a worldwide problem due to the high stability of P–O bonds.

Degrading Pesticides with Waste Product: Imidazole-Functionalized Rice Husk Catalyst for Organophosphate Detoxification

Rice husk (RH) is one the largest agricultural waste products worldwide, and rice is one of the crops that use the most pesticides. Among these, organophosphates have been of increasing concern due to their high toxicity. Herein, we report the functionalization of RH with imidazole groups (RHIMZ) to obtain sustainable catalysts from waste for organophosphate degradation. The waste-derived catalyst showed prominent catalytic activity in dephosphorylation reactions with the model substrate diethyl 2,4-dinitrophenyl phosphate (DENDPP), over 105-fold, compared to the spontaneous reaction (lifetime = 1 month). Finally, RHIMZ was also effective in degrading the pesticide Paraoxon (spontaneous lifetime = 1 million years), degrading 60% in 20 days, giving a 107-fold enhancement. Overall, the proposed approach is environmentally friendly for reusing the waste for a noble cause, i.e., degrading toxic pesticides, which is promising for designing sensors and detoxification processes.

Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency.

The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in α-esterase 7 (LcαE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcαE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization.

Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification.

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

Hybrid sol–gel bio-films: influence of synthetic parameters on behaviour and performance of entrapped His6-tagged organophosphorus hydrolase

The presented work describes the optimising of sol–gel process parameters including the aging time of the sol, the water/silane ratios, type of precursor [tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS)], and the precursor ratios using organically modified silanes [methyltrimethoxysilane (MTMOS) and 3-glycidoxypropyltrimethoxysilane (GPTMS)], for the successful entrapment of hexahistidine-tagged organophosphorus hydrolase (His6-OPH) and its applicability during organophosphate detoxification processes. The prepared bio-composite films retained enzyme activity by up to 90 % with expected differences between the starting sols’ compositions. The obtained values of KM and Vmax for TMOS/MTMOS and TEOS/GPTMS bio-composites equalled 1.5 ± 0.1, 1.4 ± 0.3 mM, 0.40 ± 0.02 and 1.8 ± 0.2 μM/s for paraoxon, respectively, thus assuming effective application of such biocatalysts during the detoxifications of organophosphorus compounds. Furthermore, the bio-composite films showed enhanced pH and temperature stability with respect to the free enzymes as they can be easily separated from the reactive media, as well as having multiple reusages. Bio-composite materials based on hexahistidine-tagged organophosphorus hydrolase were developed by enzyme encapsulation into silica sol–gel matrixes (tetramethoxysilane/methyltrimethoxysilane, tetraethoxysilane/3-glycidoxypropyltrimethoxysilane). The process of biomaterial production was optimised in several pathways and biomaterials were characterised via IR-spectroscopy, SEM-imaging, so as their catalytic efficiency in organophosphate hydrolysis.

Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp.

OPHC2 is a thermostable organophosphate (OP) hydrolase in the β-lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962- and 106-fold improvements in catalytic efficiency for methyl-parathion and ethyl-paraoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 × 10(7) -fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 Å resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPHM2 from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg(-1) ) toward methyl-parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in β-lactamase superfamily and provide efficient and robust enzymes for OP detoxification.

Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting

Paraoxonase-1 (PON1) and butyrylcholinesterase (BCHE) are natural bioscavengers of organophosphate acetylcholinesterase inhibitors in the human body, which can determine individual sensitivity to organophosphate toxicity. Interindividual differences in activity of PON1 (catalytic bioscavenger) and substrate specificity are strongly associated with the substitution of two amino acids: Leu/Met (L/M) at position 55 (rs854560) and Gln/Arg (Q/R) at position 192 (rs662). In the case of BCHE (stoichiometric bioscavenger) substitution, Ala/Thr (A/T) at position 539 produces the so-called "K-variant" of the enzyme (rs1803274). Threonine allele is often co-inherited with an atypical BCHE allele (rs1799807). The atypical variant of BCHE displays a lower affinity for cholinesterase inhibitors. Genotyping rs662 and rs1803274 single-nucleotide polymorphisms (SNP) by high-resolution melting (HRM) is facilitated by the nucleotide substitution A>G (G>A), which resulted in a changed number of hydrogen bonds in the PCR product and, consequently, shifted T m. In the case of rs854560, genotyping is complicated by the nucleotide substitution T>A, which has no significant effect on the T m of the PCR product. An addition of a small quantity of LL homozygote DNA into the reaction mixture before PCR discriminates the three genotypes by the melt curves due to different amounts of heteroduplexes formed in the LM and MM samples. HRM analysis can be applied for genotyping human rs854560, rs662, and rs1803274 SNPs.