From metabolism to comparative biochemistry of toxic organophosphorus compounds

The main stages of scientific biography of Professor Viktor Iosifovich Rozengart are exposed: his works on muscle bioenergetics, discovery of the pathway of synthesis of creatinine, his development of new concepts of pathways of metabolism of organophosphorus xenobiotics, creation of biochemical grounds of selective toxicity as well as studies in the new field, of which he was one of founders,—comparative biochemistry of toxic organophosphorus compounds.     

Acetylcholinesterase of cobra venom. Determination of concentration of active sites]

O-Nitrophenyl dimethylcarbamate and organophosphorus inhibitors O-n-propyl-p-nitrophenyl methylphosphonate, O-n-butyl-p-nitrophenyl methylphosphonate, O,O-diethyl-p-nitrophenyl phosphate, O-n-butyl-S-(beta-ethylmercaptoethyl) methylthiophosphonate, methysulphate of O,O-diethyl-S-(beta-phenyldimethylammoniumethly) thiophosphate were used in the titration of acetylcholinesterase active site concentratration in Naja naja oxiana venom. No side reactions with the acetylcholinesterase molecule as well as with other components of the venom were observed. In titration the effective concentrations of organophosphorus inhibitors with asymmetric phosphorus were 50% of their analytical concentrations, since cobra venom cholinesterase showed practically absolute stereoselectivity against the compounds.     

Studies on the Organophosphorus Insecticides

During the course of investigation on organophosphorus insecticides, many organophosphorus compounds having methyl-, methyl-chloro-, methyl-nitro-, methyl-cyano-, methyl-thiocyano-, methoxy-nitro-, acyloxy-nitro-, and chloro-cyano-phenyl groups were prepared and their biological activities tested. As the results of these studies, two compounds were found as new low toxic organophosphorus insecticides, namely, O,O-dimethyl-O- (3-methyl-4-nitro-phenyl) and O,O-dimethyl-O- (4-cyanophenyl) phosphorothioates.     

Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate.

A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity against several highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorus acid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a single polypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme has optimum activity at pH 8.5 and 50 degrees C, and it is stimulated by manganese and cobalt.     

The inhibition of locomotion of the polymorphonuclear leucocyte by organophosphorus compounds

The effects of diisopropylphosphorofluoridate (DFP) and other organophosphorus compounds on the locomotion of rabbit polymorphonuclear leucocytes have been investigated in vitro using time-lapse cinémicrography. Both phosphorylating and non-phosphorylating compounds were observed to inhibit cell locomotion, not only increasing the proportion of stationary cells, but also decreasing the velocity of those cells whose movement continued. This inhibition of locomotion occurred over the same concentration range of organophosphorus compound which was previously found to enhance the effect of leucocidin on the leucocyte. Although the inhibitory effects of low concentrations of organophosphorus compounds were partly reversible, higher concentrations produced effects which continued to increase even after the cells had been returned to normal medium. It is suggested that the supposed effect of organophosphorus compounds on chemotaxis may actually be due to the inhibition of locomotion per se, probably through the detergent properties of these compounds rather than their properties as enzyme inhibitors.     

Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His(6)-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.     

Cytogenetic effects of pesticides. II. Induction of micronuclei in mouse bone marrow by the insecticide gardona

The induction of micronuclei in mouse bone marrow by the organophosphorus insecticide gardona (also known as tetrachlorvinphos) was tested. 3 routes of administration were used for the pure insecticide: intraperitoneal, oral and dermal. The different routes of treatment with gardona caused toxicity of marrow indicated as significant increases in the percentage of polychromatic erythrocytes over that of the control. Intraperitoneal and oral treatments induced a statistically significant percentage of micronucleated PE.     

Nano-Intercalated Organophosphorus-Hydrolyzing Enzymes in Organophosphorus Antagonism

A dendritic poly(2-alkyloxazoline)-based polymer was studied as a new carrier system for the organophosphorus-hydrolyzing recombinant enzymes, organophosphorus acid anhydrolase and organophosphorus hydrolase. Paraoxon (PO) and diisopropylfluorophosphate (DFP) were used as model organophosphorus compounds. Changes in plasma cholinesterase activity were monitored. The cholinesterase activity was proportional to the concentrations of DFP or PO. Plasma cholinesterase activity was higher in animals receiving enzyme and oxime before the organophosphates than in the oxime-only pretreated groups. These studies suggest that cholinesterase activity can serve as an indicator for the in vivo protection by the nano-intercalated organophosphorus acid anhydrolase or organophosphorus hydrolase against organophosphorus intoxications. These studies represent a practical application of polymeric nano-delivery systems as enzyme carriers in drug antidotal therapy.     

Phosphorus-containing pesticide breakdown products: quantitative utilization as phosphorus sources by bacteria.

Bacteria were isolated that could utilize representatives of the following ionic phosphorus-containing breakdown products of organophosphorus pesticides as sole phosphorus sources: dialkyl phosphates, dialkyl phosphorothioates, dialkyl phosphorodithioates, alkyl arylphosphonates, alkyl arylphosphonothioates, and alkyl alkylphosphonates. Utilization of each organophosphorus compound, which was complete for 7 of 12 compounds studied, was confirmed by determination of protein yield from the amount of phosphorus source consumed. This is the first report of the utilization of an ionic dialkyl thiophosphate or dithiophosphate by microorganisms.     

A thin film electro-acoustic enzyme biosensor allowing the detection of trace organophosphorus pesticides

We report an analytical method using a thin film electro-acoustic resonator for the detection of organophosphorus pesticides. The acetylcholinesterase (AChE) enzyme was immobilized on the surface of the resonator. In the presence of organophosphorus compounds, the degree of inhibitory effect of organophosphorus compounds on the AChE activity and the concentration of pesticides were detected in real time by measuring the frequency shift of the resonator. The proposed device has a remarkably low detection limit of 1.8×10(-11)M and obvious advantages such as small size, simple operation, and integrated circuit compatibility, providing a promising tool for pesticide analysis.     

Acute fenitrothion poisoning.

A technician was accidentally exposed to the organophosphorus insecticide fenitrothion and subsequently treated with pralidoxime for the autonomic, somatic and psychiatric manifestations of intoxication. Although long-term therapy with pralidoxime is not recommended, this patient required and obtained symptomatic benefit from its prolonged use. Erythrocytic and plasma cholinesterase activities had been monitored prior to exposure and were monitored throughout treatment and following recovery. Continual monitoring of cholinesterase activities of individuals occupationally exposed to organophosphorus ester insecticides and early diagnosis are essential.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His₆-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K_m and k_cat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min⁻¹, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

Metabolic products of microorganisms 181. Chitin synthase from fungi,a test model for substances with insecticidal properties

Chitin synthase from Coprinus cinereus (Schaeff. ex Fr.) S. F. Gray (= C. lagopus sensu Buller) was used as a model for chitin synthase from insects. The effect of dimilin (difluorobenzuron), captan (trichloromethylsulfonyl fungicide), kitazin P (organophosphorus ester fungicide) and parathion (organophosphorus insecticide) on the fungal enzyme was compared with the effect of nikkomycin (nucleoside-peptide antibiotic).Metabolic products of microorganisms. 180. M. Brufani, L. Celai, W. Keller-Schierlein, E. Pretsch: Revised structure of naphthomycin. J. Antibiot. (Tokyo) (in press)     

Structural requirements for the inhibition of human monocyte carboxylesterase by organophosphorus compounds

Human blood monocyte carboxylesterase (CBE) is inhibited by a variety of organophosphorus compounds including arylphosphates and arylphosphites and some alkylphosphites. Triphenyl phosphate and triphenyl phosphite with K_i values of 8 × 10⁻⁹ M and 4.8 × 10⁻⁸ M, respectively, are the most potent inhibitors of this enzyme evaluated by this study. The arylphosphates vary in their capacity to inhibit carboxylesterase activity. Diphenyl phosphate with its strong negative charge is not a potent inhibitor (K_i = 1 × 10⁻⁴ M), whereas if its negative charge is neutralized, as in diphenyl methyl phosphate, its capacity to inhibit carboxylesterase is significantly increased. Compounds with increased bulk, such as trinaphthyl phosphate, only inhibit the enzyme at concentrations of 10⁻⁵ M or greater. Arylphosphites have inhibitory capacities similar to the arylphosphates. Alkylphosphites (tributyl phosphite/triethyl phosphite) inhibit carboxylesterase activity, whereas alkylphosphates (tributyl phosphate/triethyl phosphate) have no inhibitory effect. Arylphosphines and arylphosphine oxides do not inhibit carboxylesterase activity. This study demonstrates that organophosphates and organophosphites are relatively effective inhibitors of human monocyte CBE activity with the exception of the alkylphosphates which have no inhibitory activity. We conclude that molecular bulk and charge have a significant role in determining the potency of organophosphorus inhibitors of monocyte CBE. The observed variations in the degree of esterase inhibition by organophosphorus compounds as well as the differences in the pathological expression of neuropathic disorders associated with such chemicals suggest that different esterase enzymes derived from the family of esterase genes may mediate the different neuropathies observed with organophosphorus exposures. Such data also provide the rationale for the kinetic analyses of esterases and the design of non-toxic organophosphorus compounds with low or no monocyte CBE inhibitory capacity to reduce the potential of these commonly used chemicals for human toxicity.     

Microbial degradation of organophosphorus compounds

Synthetic organophosphorus compounds are used as pesticides, plasticizers, air fuel ingredients and chemical warfare agents. Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of world-wide insecticide sales. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. Several reports suggest that a wide range of water and terrestrial ecosystems may be contaminated with organophosphorus compounds. These compounds possess high mammalian toxicity and it is therefore essential to remove them from the environments. In addition, about 200,000 metric tons of nerve (chemical warfare) agents have to be destroyed world-wide under Chemical Weapons Convention (1993). Bioremediation can offer an efficient and cheap option for decontamination of polluted ecosystems and destruction of nerve agents. The first micro-organism that could degrade organophosphorus compounds was isolated in 1973 and identified as Flavobacterium sp. Since then several bacterial and a few fungal species have been isolated which can degrade a wide range of organophosphorus compounds in liquid cultures and soil systems. The biochemistry of organophosphorus compound degradation by most of the bacteria seems to be identical, in which a structurally similar enzyme called organophosphate hydrolase or phosphotriesterase catalyzes the first step of the degradation. organophosphate hydrolase encoding gene opd (organophosphate degrading) gene has been isolated from geographically different regions and taxonomically different species. This gene has been sequenced, cloned in different organisms, and altered for better activity and stability. Recently, genes with similar function but different sequences have also been isolated and characterized. Engineered microorganisms have been tested for their ability to degrade different organophosphorus pollutants, including nerve agents. In this article, we review and propose pathways for degradation of some organophosphorus compounds by microorganisms. Isolation, characterization, utilization and manipulation of the major detoxifying enzymes and the molecular basis of degradation are discussed. The major achievements and technological advancements towards bioremediation of organophosphorus compounds, limitations of available technologies and future challenge are also discussed.     

Interaction of insecticides with lipid membranes.

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10(-5)--10(-4) M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K+: parathion greater than 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) approximately aldrin greater than malathion greater than lindane. The degree of effectiveness for X-537A-induced permeation of Ca2+ was the following: aldrin greater than or equal to DDT greater than parathion greater than malathion greater than lindane. The organophosphorus compound, ethyl azinphos (10(-4) M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes. It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

Prenatal exposure to pesticides: analysis of human placental acetylcholinesterase, glutathione S-transferase and catalase as biomarkers of effect.

Pre- and perinatal exposure to pesticides is deleterious on foetal and neonatal development, but information regarding possible effects on environmental low-dose exposure to pesticides is scarce. Most epidemiological studies of the health effect of pesticides have been based on self-reported information. However, detailed information on past pesticide use is difficult to reconstruct. This is a current study conducted among pregnant mothers attending a delivery care and perinatal programme at a public hospital. The study investigates biomarkers of early effects in placentas from women living in an area with an intensive use of pesticides in the northern part of Patagonia, province of Río Negro, Argentina, and it assesses the consistency of the information provided by self-reports. The study confirms that placental acetylcholinesterase and catalase activities are significantly associated with periods of organophosphorus pesticides application, while glutathione S-transferase is not affected. We found a positive correlation between environmental exposure to organophosphorus pesticides and carbamate insecticides and newborn head circumference. The findings provide a further indication of a link between placenta acetylcholinesterase and catalase activity and prenatal exposure to pesticides in population studies. Both placenta enzymes may be used as biomarkers in health surveillance programmes for early diagnosis of exposure related alterations produced by organophosphorus pesticides and carbamate pesticides.