Detoxification Of Organophosphorus Compounds Research Articles

Enhancing organophosphate hydrolase efficacy via protein engineering and immobilization strategies

Organophosphorus compounds (OPs), developed as pesticides and chemical warfare agents, are extremely toxic chemicals that pose a public health risk. Of the different detoxification strategies, organophosphate-hydrolyzing enzymes have attracted much attention, providing a potential route for detoxifying those exposed to OPs. Phosphotriesterase (PTE), also known as organophosphate hydrolase (OPH), is one such enzyme that has been extensively studied as a catalytic bioscavenger. In this review, we will discuss the protein engineering of PTE aimed toward improving the activity and stability of the enzyme. In order to make enzyme utilization in OP detoxification more favorable, enzyme immobilization provides an effective means to increase enzyme activity and stability. Here, we present several such strategies that enhance the storage and operational stability of PTE/OPH.

EFFECTS OF FATTY ACIDS ON BINDING AND ESTERASE ACTIVITY OF ALBUMIN TOWARDS ORGANOPHOSPHORUS COMPOUNDS ACCORDING TO MOLECULAR MODELING APPROACH

One of the urgent tasks of clinical toxicology is the development of therapy aimed at stoichiometric and/or catalytic detoxification of organophosphorus compounds in the bloodstream, which will prevent the poison’s entering the neuromuscular and neuronal synapses and help to avoid irreversible consequences of poisoning. An auxiliary option for the detoxification of organophosphorus compounds in the bloodstream may be a directed effect on albumin, the main transport protein of the blood, by means of molecules modulating its binding and/or esterase properties. The aim of the present study is to evaluate the effect of fatty acids on the binding and esterase activity of human albumin to organophosphorus compounds by molecular modeling methods on the example of paroxone and oleic acid. According to the data obtained, an increased concentration of fatty acids in the blood reduces the likelihood of paraoxon binding to albumin and pseudo-esterase reaction.

Asymmetric biodegradation of the nerve agents Sarin and VX by human dUTPase: chemometrics, molecular docking and hybrid QM/MM calculations

Organophosphorus compounds (OP) nerve agents are among the most toxic chemical substances known. Their toxicity is due to their ability to bind to acetylcholinesterase. Currently, some enzymes, such as phosphotriesterase, human serum paraoxonase 1 and diisopropyl fluorophosphatase, capable of degrading OP, have been characterized. Regarding the importance of bioremediation methods for detoxication of OP, this work aims to study the interaction modes between the human human deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and Sarin and VX, considering their Rp and Sp enantiomers, to evaluate the asymmetric catalysis of those compounds. In previous work, this enzyme has shown good potential to degrade phosphotriesters, and based on this characteristic, we have applied the human dUTPase to the OP degradation. Molecular docking, chemometrics and mixed quantum and molecular mechanics calculations have been employed, showing a good interaction between dUTPase and OP. Two possible reaction mechanisms were tested, and according to our theoretical results, the catalytic degradation of OP by dUTPase can take place via both mechanisms, beyond being stereoselective, that is, dUTPase cleaves one enantiomer preferentially in relation to other. Chemometric techniques provided excellent assistance for performing this theoretical investigation. The dUTPase study shows importance by the fact of it being a human enzyme.Communicated by Ramaswamy H. Sarma

Modulation of the albumin–paraoxon interaction sites by fatty acids: Analysis by the molecular modeling methods

It is known that albumin can break the ester bonds in organophosphorus compounds (OPs). Amino acids responsible for esterase and pseudoesterase activity of albumin towards OPs are still not determined. It is assumed that Sudlow’s site I with residue Tyr150 exhibits the “true” esterase activity; and Sudlow’s site II containing residue Tyr411, a pseudoesterase one. Binding of fatty acids to albumin affects the efficiency of its interaction with xenobiotics; however, the effect of fatty acids on the interaction of albumin with OPs was not studied. The purpose of this work was to study the interaction of OPs with potential sites of albumin enzymatic activity and to examine the effect of fatty acids on the efficiency of such interaction using the molecular modeling methods by the example of paraoxon, a known inhibitor of acetylcholinesterase, and oleic acid. The structures of the protein complexes with paraoxon and oleic acid were determined by the molecular docking procedure; the conformational changes were calculated by the molecular dynamics method. It has been shown that sorption of oleic acid in one of the fatty acid-binding sites leads to the conformational changes in Sudlow’s sites I and II due to a “reversal” of the side chains of Arg410 and Arg257 residues by 90°. It has been found that this change in geometry reduces the affinity of Sudlow’s site I and increases the Sudlow’s site II affinity to paraoxon. The amino acid residue Ser193, which was previously identified as a site of possible albumin esterase activity, is not able to bind paraoxon efficiently. It is assumed that its activity can be affected by the interaction of the oleic acid molecules with other fatty acid-binding sites. It is hypothesized that the lesser toxicity of paraoxon compared to soman may be associated not only with its lower inhibitory activity against cholinesterases, but also with the increased affinity of paraoxon to albumin. It was concluded that albumin may serve as an alternative means of OP detoxification.

Computational enzymology for degradation of chemical warfare agents: promising technologies for remediation processes.

Chemical weapons are a major worldwide problem, since they are inexpensive, easy to produce on a large scale and difficult to detect and control. Among the chemical warfare agents, we can highlight the organophosphorus compounds (OP), which contain the phosphorus element and that have a large number of applications. They affect the central nervous system and can lead to death, so there are a lot of works in order to design new effective antidotes for the intoxication caused by them. The standard treatment includes the use of an anticholinergic combined to a central nervous system depressor and an oxime. Oximes are compounds that reactivate Acetylcholinesterase (AChE), a regulatory enzyme responsible for the transmission of nerve impulses, which is one of the molecular targets most vulnerable to neurotoxic agents. Increasingly, enzymatic treatment becomes a promising alternative; therefore, other enzymes have been studied for the OP degradation function, such as phosphotriesterase (PTE) from bacteria, human serum paraoxonase 1 (HssPON1) and diisopropyl fluorophosphatase (DFPase) that showed significant performances in OP detoxification. The understanding of mechanisms by which enzymes act is of extreme importance for the projection of antidotes for warfare agents, and computational chemistry comes to aid and reduce the time and costs of the process. Molecular Docking, Molecular Dynamics and QM/MM (quantum-mechanics/molecular-mechanics) are techniques used to investigate the molecular interactions between ligands and proteins.

New and highly active microbial phosphotriesterase sources.

Many toxic insecticides used worldwide as well as some chemical warfare agents are phosphotriester derivatives. Therefore, detoxification of organophosphorus compounds has become the subject of many studies and in particular bioremediation, based on the phosphotriesterase catalysed hydrolysis of these compounds, has shown to be an effective and ecological methodology. In order to identify new bacterial phosphotriesterases, a simple and sensitive fluorimetric screening method on solid media was employed that allowed the selection of six strains with phosphotriesterase activity. Since pH and temperature are important parameters for bioremediation of contaminated soils and waters, the influence of these variables on the rate of the enzymatic hydrolysis was assessed. This study afforded notable results, being the most remarkable one the increased activity exhibited by Nocardia asteroides and Streptomyces setonii strains at 50°C, 7 and 30 times higher than at 30°C, respectively. Compared with the results obtained with Brevundimonas diminuta, whose activity is usually considered as reference, an increase of 26 and 75 times is observed, respectively.

Biochemical basis for hydrolysis of organophosphorus by a marine bacterial prolidase

Abstract Extensive application of synthesized organophosphorus compounds (OPs) leads to pollutant accumulation and enhanced eco-toxicity. Hydrolysis of phosphotriester bonds catalyzed by evolved microbial enzymes is a key step for detoxification of OPs. Here, a new marine bacterial prolidase OPAA4301 exhibiting promiscuous phosphotriesterase activity was isolated and systematically characterized. The homo-tetrameric enzyme OPAA4301 can catalyze the hydrolysis of both amido bond and phosphotriester bond. Manganese ions were observed to be essential for its catalytic integrity, and in vitro substitution of manganese ions by different metal cofactors led to decreased activity. We also revealed cooperation pattern of metal ligands and substrate-binding residues on OP hydrolysis by mutational analysis. Metal-binding sites together with Arg418 in the large-binding pocket of the enzyme were found to be indispensable for catalytic ability. Substitution mutation of small- and large-binding pocket residues caused significant variation in phosphotriesterase activity, and leaving group sites appeared to be involved in the catalytic process as substrate affinity regulators. Our study gave an overall biochemical understanding on the organophosphorus hydrolysis pattern of the newly identified marine bacterial prolidase and provided ideas for protein engineering to expand its application in the bioremediation field.

Elimination pathways of cyclosarin (GF) mediated by β-cyclodextrin in vitro: Pharmacokinetic and toxicokinetic aspects

Cyclodextrins (CD) are promising small molecular scavengers showing favourable degradation of extremely toxic organophosphorus compounds (OP) such as tabun (GA), soman (GD) or cyclosarin (GF). For β-CD derivatives as potential OP antidotes with low intrinsic toxicity it is of great interest to completely understand the modes of interaction of both compounds in terms of OP detoxification. The mechanisms of CD action are not completely understood which prompted us to investigate the interactions of GF and β-cyclodextrin (β-CD) as model compounds. Using positive electrospray ionization mass spectrometry (ESI/MS), the formation of covalent conjugates of β-CD with O-cyclohexylmethylphosphonate (CHMP) residue was detected for the first time and was examined in vitro. With a newly developed LC–MS method the formation of O-cyclohexylmethylphosphonic acid (CHMPA) (i.e. GF hydrolysis) and covalent CHMP-β-CD conjugates was analyzed. Compared to water, tris(hydroxymethyl)aminomethane (TRIS) reduced the formation of covalent conjugates but amplified formation of CHMPA. Depending on experimental conditions the degradation of GF by β-CD may be preferably catalytic or stoichiometric. For illustrating different possible reaction pathways a scheme was established that could support the idea of β-CD acting as an artificial enzyme. These results provide an important insight into the β-CD mediated detoxification pathways of GF.

English

Organophosphorus compounds (OPs) are widely used as pesticides and chemical warfare agents (CWAs). These synthetic compounds reside in the environment and cause serious pollution problems. Organophosphorus compounds can be detoxified rapidly by hydrolysis on exposure to environment, which are liable to be influenced by abiotic and biotic factors. Microorganisms isolated from different niches are the predominant entities in the biosphere with an exceptional ability to metabolize various OPs for their growth. The focus of this work is organophosphorus hydrolase (OPH, E.C. 8.1.3.1), which catalyzes the hydrolysis of many organophosphorus compounds and greatly reduces the toxicity and even can completely mineralize them. Several OPH enzymes, including o-phenylenediamine dihydrochloride (OPD), methyl parathion hydrolase (MPH) mevalonate pyrophosphate decarboxylase (MPD) etc, have been identified which accomplish hydrolysis for specific classes of organophosphorus compounds. The functional gene encoding OPH protein have been cloned, expressed and purified in both prokaryotic and eukaryocyte expression vector. To increase the enzyme activity or enhance their broad-spectrum property, the wild activated OPH was used to modify activated sites by the chemical modification of specific amino acid residues with the use of appropriately designed coenzyme analogs. The applications of the functional strains and OPH enzymes in bioremediation of OPs pollutants included: (i) Bioremediation: the technologies can be generally classified as in situ or ex situ; (ii) immobilization: an immobilized enzyme is the OPH that is attached to an inert, insoluble material such as calcium alginate and agar; immobilized whole cell, the target cell is the functional strains that is capable of degrading special OPs; (iii) construction of genetically engineering bacteria. A versatile genetically engineering bacterium that gained more functional genes from different sources was constructed by gene engineering and enzyme engineering, showing a creative and promising application in OPs detoxification. Key words: Organophosphorus compounds, biodegradation, bioremediation, oph, genetically engineering.

Biochemical, Environmental, and Genetic Factors Associated with Paraoxonase (PON1) Activity

Paraoxonase (PON1, EC 3.1.8.1) is tightly linked to high-density lipoprotein (HDL) and shows significant correlation with apo AI concentrations. It plays an important role in functions such as protection against vascular disease (e.g., atherosclerosis) through its inhibition of low-density lipoprotein (LDL) oxidation and detoxification of organophosphorus compounds by hydrolyzing the bioactive oxons (Mackness et al. 1991). The activity of PON1 is modulated by genetic and environmental factors. Two coding region polymorphisms of the PON1 gene result in amino acid substitutions at position 192 (PON1 Q192R, glutamine to arginine) and at position 55 (PON1 L55 M, leucine to methionine). PON1 Q192R has been reported to account for 73–76% of the variation in hydrolysis activity of paraoxon (paraoxonase activity) and 12–25% of the variation in activity for the hydrolysis of diazoxon (diazonase activity) in vitro (Jarvik et al. 2000). The L55 M polymorphism also contributes to PON1 protein stability and activity (Leviev et al. 2001). PON1 polymorphisms have been studied in many populations of the world; the frequency of the low-activity

The role of paraoxonase polymorphisms in the induction of micronucleus in paraoxon‐treated human lymphocytes

Human paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme that has a role in the detoxification of organophosphorus compounds by hydrolyzing the bioactive oxons. PON1 polymorphims are responsible, at least in part, for the variation in the catalytic activity and expression of the enzyme and have been associated with susceptibility to organophosphorus pesticide toxicity, mainly neurotoxicity. The aim of this study was to determine whether paraoxon induced micronuclei and to examine the role of PON1 polymorphism in paraoxon's genotoxic potential. First, dose finding cytogenetic experiments were performed on lymphocyte cultures from three donors and a range of paraoxon concentration (1-25 microM) were tested. In a second set of experiments, 5 microM paraoxon was added to blood cultures of 11 donors with two different PON1 haplotypes (PON T(-108)M(55)Q(192) with low activity and haplotype PON C(-108)L(55) R(192) with high activity, referred to as PON1QQ and as PON1 RR, respectively). Because PON1 is present in blood, the effect of adding 5 microM paraoxon and 70 microl of autologous plasma to lymphocyte cultures also was examined. Paraoxon had no effect on cell viability, but caused a significant dose-dependent increase in MN frequency. The basal MN frequencies were similar on QQ and RR genotypes. A significant difference was observed in the MN frequency only in lymphocytes from individuals with the QQ genotype treated with 5 microM paraoxon and the autologous plasma did not modify these effects. The results obtained in this study suggest that PON1 genotype might have an important role in the identification of individuals at risk for cancer development due to occupational exposure to pesticides.

Genetic variability in the cytochrome P450–paraoxonase 1 (PON1) pathway for detoxication of organophosphorus compounds

Genetic variability in the cytochrome P450–paraoxonase 1 (PON1) pathway for detoxication of organophosphorus compounds

Bioscavengers for the protection of humans against organophosphate toxicity

Current antidotes for organophosphorus compounds (OP) poisoning consist of a combination of pretreatment with carbamates (pyridostigmine bromide), to protect acetylcholinesterase (AChE) from irreversible inhibition by OP compounds, and post-exposure therapy with anti-cholinergic drugs (atropine sulfate) to counteract the effects of excess acetylcholine and oximes (e.g., 2-PAM chloride) to reactivate OP-inhibited AChE. These antidotes are effective in preventing lethality from OP poisoning, but they do not prevent post-exposure incapacitation, convulsions, seizures, performance decrements, or in many cases permanent brain damage. These symptoms are commonly observed in experimental animals and are likely to occur in humans. The problems intrinsic to these antidotes stimulated attempts to develop a single protective drug, itself devoid of pharmacological effects, which would provide protection against the lethality of OP compounds and prevent post-exposure incapacitation. One approach is the use of enzymes such as cholinesterases (ChEs), β-esterases in general, as single pretreatment drugs to sequester highly toxic OP anti-ChEs before they reach their physiological targets. This approach turns the irreversible nature of the OP: ChE interaction from disadvantage to an advantage; instead of focusing on OP as an anti-ChE, one can use ChE as an anti-OP. Using this approach, it was shown that administration of fetal bovine serum AChE (FBSAChE) or equine serum butyrylcholinesterase (EqBChE) or human serum BChE (HuBChE) protected the animals from multiple LD 50s of a variety of highly toxic OPs without any toxic effects or performance decrements. The bioscavengers that have been explored to date for the detoxification of OPs fall into three categories: (A) those that can catalytically hydrolyze OPs and thus render them non-toxic, such as OP hydrolase and OP anhydrase; (B) those that stoichiometrically bind to OPs, that is, 1 mol of enzyme neutralizes one or 2 mol of OP inactivating both, such as ChEs and related enzymes; and (C) and those generally termed as “pseudo catalytic”, e.g., a combination of ChE and an oxime pre-treatment such that the catalytic activity of OP-inhibited ChE can rapidly and continuously be restored in the presence of an oxime. Since the biochemical mechanism underlying prophylaxis by exogenous esterases such as ChEs is established and tested in several animal species, including non-human primates, this concept should allow a reliable extrapolation of results from animal experiments to human application. Having being extensively investigated by several groups, plasma derived HuBChE is judged to be the most suitable bioscavenger for its advancement for human use. The program is being developed at the present time for conducting a safety clinical trial in human volunteers. Several other candidate bioscavengers will follow; e.g., recombinant HuBChE expressed in the milk of transgenic goats, pseudo catalytic scavenger(s), e.g., a combination of ChE and oxime, and possibly PON 1 as a catalytic scavenger in the future.

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5–4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0–5.5-times higher compared to the immobilized form mainly due to the lower Km values. With constant 55–60% humidity the biocatalyst retained 77% and 67–70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.

Nuevas perspectivas en los tratamientos de intoxicaciones por insecticidas organofosforados y agentes nerviosos de guerra

Organophosphorus compounds are worldwide employed as insecticides and are yearly responsible of several millions of poisonings. The chemical structure of most of the warfare nerve agents also corresponds with an organophosphorus compound. Organophosphorus insecticides and warfare nerve agents exert their main toxicological effects through inhibition of acetylcholinesterase. Current treatments of patients poisoned with organophosphorus compounds include atropine (in order to protect muscarinic receptors), oximes (in order to accelerate the reactivation of the inhibited acetylcholinesterase) and benzodiazepines (in order to avoid convulsions). The administration of phosphotriesterases (enzymes involved in the detoxication of organophosphorus compounds through hydrolysis) is a very effective treatment against poisonings by organophosphorus insecticides and warfare nerve agents. There are experimental preventive treatments based on the simultaneous administration of carbamates and certain antimuscarinic drugs, different from atropine, which notably improve the efficacy of the classical treatments applied after poisonings by warfare nerve agents. The treatments based in the administration of phosphotriesterases might be the response to the call of the World Health Organization for searching new treatments with capability to reduce the high mortality recorded in the cases of poisonings by organophosphorus compounds. These treatments can be applied in a preventive way without the intrinsic neurotoxicity associated to the preventive treatments based on carbamates and antimuscarinic drugs. Therefore, these treatments are specially interesting for people susceptible to suffer severe exposures, i.e. sprayers in the farms.

Biotransformation of organophosphorus compounds

This study reviews current understanding of mechanisms of biotransformation of organophosphorus compounds (OPC). The first part of this article covers chemical aspects of biotransformation describing reactions that lead to activation or detoxication of OPC. The second part explains biochemical mechanisms of biotransformation describing the role of enzymes that are involved in this process. Among them are the enzymes that take part in metabolic activation of OPC such as cytochrome P450 system, NADPH–cytochrome P450 reductase and flavin-containing monooxygenases. Among enzymes participating in detoxication of OPC, the role of phosphoric triester hydrolases, carboxylesterases and glutathione redox system is explained. This article also deals with other aspects of detoxication of OPC such as protein binding and the role of tissue depots for these compounds.

Molecular cloning and expression of rat lung carboxylesterase and its potential role in the detoxification of organophosphorus compounds.

The 1,839-base pair complementary DNA (cDNA) for rat lung carboxylesterase was cloned by reverse transcriptase polymerase chain reaction from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to be similar to hepatic CEH, pI 6.1 esterase, and hydrolase A. In Northern blot analysis, the cDNA hybridized with a single band from lung messenger RNA (mRNA). The 1.7-kb coding sequence, predicting a 62-kD protein, was transfected into COS-7 cells and Chinese hamster ovary (CHO) cells. Expression in COS-7 and CHO cells was accompanied by 4- and 3.2-fold increases in carboxylesterase activity (hydrolysis of p-nitrophenyl acetate), respectively. Unlike the hepatic CEH, the expressed lung carboxylesterase described here did not hydrolyze cholesterol esters. In situ hybridization experiments localized the lung carboxylesterase mRNA to the airway epithelium. The organophosphorus compound phosphoric acid diethyl 4-nitrophenyl ester, paraoxon, completely inhibited this lung carboxylesterase, placing it in the family of B esterases by this criterion.

The Role of Phosphotriesterases in the Detoxication of Organophosphorus Compounds

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 phosphotriesterases and susceptibility of the species to the toxic effects of organophosphorus compounds. Thus, mammals that possess 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 are currently subject to study.

Interaction of organophosphorus compounds with carboxylesterases in the rat.

Carboxylesterases (CarbE) are involved in detoxication of organophosphorus compounds (OPC) through two mechanisms: hydrolysis of ester bonds in OPC which contain them and binding of OPC at the active site of CarbE which reduces the amount of OPC available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was undertaken in order to contribute to better understanding of the role of CarbE in detoxication of OPC. The results obtained have shown that inhibitory potency (I50) of dichlorvos, sarin and soman towards rat liver CarbE was 0.2 microM, 0.5 microM and 4.5 microM, respectively, for 20-min incubation at 25 degrees C. Second-order rate constants (k(a)) for liver CarbE inhibition were 2.3 x 10(5) M-1 min-1, 6.9 x 10(4) M-1 min-1 and 1.1 x 10(4) M-1 min-1 for dichlorvos, sarin and soman, respectively. The corresponding values for plasma CarbE could not be calculated because of dominant spontaneous reactivation of inhibited CarbE. CarbE inhibited with these OPC in vitro spontaneously reactivate with half-times of 18, 143 and 497 min for sarin, dichlorvos and soman in plasma and 111, 163 and 297 min for sarin, soman and dichlorvos in liver, respectively. These results were also confirmed in experiments in vivo in which rats were subcutaneously treated with 0.5 LD50 of these agents. The half-times of spontaneous reactivation of rat plasma CarbE in vivo were 1.2, 2.0 and 2.7 h for dichlorvos, sarin and soman, respectively. These findings have changed current understanding of the mechanism of interaction of CarbE with OPC and involvement of the enzymes in detoxication of OPC, suggesting an active and important role of the enzymes in metabolic conversions of OPC to their less toxic metabolites.