P-nitrophenyl Phosphorothioate Research Articles

The interaction of methyl-parathion with serum and albumin of the neo-tropical fish Piaractus mesopotamicus

The interaction of methyl-parathion with serum and albumin of pacu, Piaractus mesopotamicus, was studied, using the fluorescence quenching technique. Pacu is a neo-tropical fish specie inhabitant of rivers from western regions of Brazil. Methyl-parathion ( O, O-dimethyl O- p-nitrophenyl phosphorothioate) is an organophosphorous pesticide still used in agriculture and fish farming in many countries. The quenching of fluorescence can be mathematically expressed by the Stern–Volmer equation to calculate quenching constants. Stern–Volmer curves analysis is able to give important information about the pesticide–albumin interaction. Our results showed that the serum quenching reached 10% when the molar ratio of pesticide/albumin was about 7:1 for the three temperatures of the experiment. For the pure albumin quenching of 10%, methyl-parathion concentrations were 6, 7 and 9 times higher than albumin at 20, 25 and 30 °C, respectively. The calculated Stern–Volmer constants at 25 °C were 9.73×10 3(±4.9×10 2) M −1 for serum and 9.20×10 3(±2.0×10 2) M −1 for albumin. It was observed that albumin quenching is the phenomenon contributing to the quenching of the pacu serum fluorescence for methyl-parathion concentration lower than 10 μM, suggesting that the protein is the most important carrier for the pesticide in serum.

Comparative studies on chlorpyrifos and methyl parathion induced oxidative stress in different parts of rat brain: Attenuation by antioxidant vitamins

Organophosphate (OP) pesticides are among the most widely used synthetic chemicals for controlling a wide variety of pests. Chlorpyrifos ( o, o′-diethyl- o-3,5,6-trichloro-2-pyridyl phosphorothionate, CPF) is among the leading OP pesticides used extensively throughout the world including India while methyl parathion ( o, o-di methyl- o- p-nitrophenyl phosphorothioate, MPT) another OP compound, widely used as insecticide and acaricide to control many biting or sucking pests of agricultural crops. Present study was carried out to compare the chronic toxicity of CPF and MPT, their potential to generate oxidative stress and ameliorating effects of antioxidant vitamins in brain of rats. Results of the present study clearly demonstrated that the oral exposure of CPF or MPT, generated oxidative stress in different parts of rat brain consequently accumulating malondialdehyde (MDA) and 4-hydroxynonanal (4HNE), the two major end products of lipid peroxidation, in all the three brain regions i.e. fore-, mid- and hind-brain. The levels of hydrogen peroxide (H 2O 2) were also increased in all the three brain regions when compared with control. CPF and MPT exposure caused decrease in the levels of reduced glutathione (GSH) and increase in the levels of oxidized glutathione (GSSG) in all the three brain regions. The increase in the levels of MDA, 4HNE, H 2O 2 and GSSG was less pronounced when CPF or MPT was given to the rats fed with a mixture of vitamin A, E and C. The present findings clearly show that oral intake of a mixture of vitamin A, E and C protects the rats from MPT or CPF induced oxidative stress and suggest that this treatment alleviates the toxicity of these pesticides to a greater extent.

Thermodynamic Origin of the Increased Rate of Hydrolysis of Phosphate and Phosphorothioate Esters in DMSO/Water Mixtures

The hydrolysis rates of the dianions of phosphate and phosphorothioate esters are substantially accelerated by the addition of polar aprotic solvents such as DMSO and acetonitrile. The activation barrier DeltaG is smaller due to a lower enthalpy of activation. The enthalpy of transfer of p-nitrophenyl phosphate (pNPP) and p-nitrophenyl phosphorothioate (pNPPT), from water to 0.6 (mol) aq DMSO (60 mol % water in DMSO) were measured calorimetrically. The enthalpies of activation for the hydrolysis reactions in the two solvents permitted the calculation of the enthalpy of transfer of the transition states. This transfer is thermodynamically favorable for both the reactants and the transition states but is more favorable for the transition states. In the case of pNPP, the enthalpy of transfer of the reactant is -23.9 kcal/mol, compared to -28.3 for the transition state. The difference is greater for pNPPT, where the enthalpy of transfer of the reactant is -23.2 kcal/mol and that for the transition state is -35.3. The results show that the reduced enthalpies of activation in both hydrolysis reactions arise not from a destabilization of the reactants in the mixed solvent, but from the fact that the enthalpy of transfer of the transition states to the mixed solvent is significantly more negative than the enthalpy of transfer of the reactants.

Photocatalytic degradation of methyl parathion: Reaction pathways and intermediate reaction products

The photocatalytic degradation of O, O-dimethyl O- p-nitrophenyl phosphorothioate (common name methyl parathion) in aqueous TiO 2 suspensions is reported in this study. The aim of this article is to identify the intermediate organic reaction products using a number of analytical techniques, such as UV–vis, HPLC, GC–MS and 1H NMR. Total organic carbon (TOC) and the concentration of inorganic ions in the reaction mixture, as a function of reaction time, are also measured to monitor the mineralization of organic chemical species. Under both, acidic and basic conditions, the primary product is methyl paraoxon, which eventually is degraded to 4-nitrophenol. This aromatic compound is mainly transformed into hydroquinone, which is later oxidized to 1,2,4-benzenetriol. GC–MS studies show that this aromatic triol is preferentially converted via an oxidative pathway into 5-hydroxymethyl-5H-furfuran-2-one. Under basic conditions, methyl parathion is converted via a reaction pathway involving methyl paraoxon, 4-nitrophenol, hydroquinone and aliphatic acids with the resulting species being refractory to be further oxidized and mineralized. On the other hand, under controlled acidic conditions, methyl parathion and various intermediates quickly degrade in a rather fast oxidation process.

The Thermodynamics of Phosphate versus Phosphorothioate Ester Hydrolysis.

Phosphorothioate esters are phosphate esters in which one of the nonbridging oxygen atoms has been replaced by sulfur. In the comparative hydrolysis reactions of phosphorothioate and phosphate esters, the sulfur substitution accelerates the rates of the monoesters while slowing the rates of diesters and of triesters. Previously measured enthalpies and entropies of activation for the hydrolysis reactions of the monoesters, p-nitrophenyl phosphate and p-nitrophenyl phosphorothioate, were compared to the activation parameters measured herein for the diesters, ethyl p-nitrophenyl phosphate and ethyl p-nitrophenyl phosphorothioate, and the triesters, diethyl p-nitrophenyl phosphate and diethyl p-nitrophenyl phosphorothioate. A consistent trend of a greater DeltaH++ for the phosphorothioate analogue was found in all three classes of ester. In the monoester case, a more positive DeltaS++ arising from a mechanistic difference (D(N) + A(N) for the phosphorothioate versus A(N)D(N) for the phosphate) compensates, resulting in a lower DeltaG++ for the phosphorothioate monoester. Spectroscopic investigations indicate there is no significant difference in bond order to the leaving group in phosphates, as compared to their phosphorothioate analogues, ruling this out as a contribution to the consistently higher enthalpies of activation.

The Thermodynamics of Phosphate versus Phosphorothioate Ester Hydrolysis

Phosphorothioate esters are phosphate esters in which one of the nonbridging oxygen atoms has been replaced by sulfur. In the comparative hydrolysis reactions of phosphorothioate and phosphate esters, the sulfur substitution accelerates the rates of the monoesters while slowing the rates of diesters and of triesters. Previously measured enthalpies and entropies of activation for the hydrolysis reactions of the monoesters, p-nitrophenyl phosphate and p-nitrophenyl phosphorothioate, were compared to the activation parameters measured herein for the diesters, ethyl p-nitrophenyl phosphate and ethyl p-nitrophenyl phosphorothioate, and the triesters, diethyl p-nitrophenyl phosphate and diethyl p-nitrophenyl phosphorothioate. A consistent trend of a greater DeltaH++ for the phosphorothioate analogue was found in all three classes of ester. In the monoester case, a more positive DeltaS++ arising from a mechanistic difference (D(N) + A(N) for the phosphorothioate versus A(N)D(N) for the phosphate) compensates, resulting in a lower DeltaG++ for the phosphorothioate monoester. Spectroscopic investigations indicate there is no significant difference in bond order to the leaving group in phosphates, as compared to their phosphorothioate analogues, ruling this out as a contribution to the consistently higher enthalpies of activation.

Methyl parathion interaction with human and bovine serum albumin

Methyl parathion (MP; O, O-dimethyl O- p-nitrophenyl phosphorothioate) is an organophosphorous compound still largely used in agriculture and fish hatcheries. This pesticide is not quite selective and is potentially toxic for both vertebrates and invertebrates. Its mechanism of acute toxicity is the inhibition of the enzyme acetylcholinesterase in nervous tissue. Binding of pesticides to plasma proteins is one of many factors that influence their distribution and elimination. The free concentration available for toxic action can be effectively reduced for pesticides with high binding to plasma proteins, although the affinity of pesticides to plasma proteins is often lower than for the enzyme targets. Several different transport proteins exist in blood plasma, but albumin only is able to bind a wide diversity of xenobiotics reversibly with high affinity. It was already known that parathion (ethyl parathion) exhibits a high affinity to human and bovine serum albumins. We studied interactions of methyl parathion with these albumins by using fluorescence quenching techniques. We selectively excited the fluorescence of tryptophan residues with a 290 nm wavelength light, and observed quenching by titrating human and bovine serum albumin solutions with methyl parathion. Stern–Volmer graphs were plotted and quenching constants were estimated. Our results pointed to the formation of complexes of methyl parathion with albumins. Association constants at 25 °C were 3.07×10 4 (1.2×10 3) M −1 for human serum albumin, and 1.96×10 4 (±4.5×10 2) M −1 for bovine serum albumin. At 37 °C, they were 1.08×10 4 (±2.0×10 2) M −1 for human serum albumin, and 8.16×10 3 (±1.9×10 2) M −1 for bovine serum albumin. Results also suggest that the primary binding site for methyl parathion on albumin is close to tryptophan residues 214 of human serum albumin and 212 of bovine serum albumin.

Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases.

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

The role of metabolism in determining susceptibility to parathion toxicity in man

Human liver microsomes ( n=16) activated parathion ( O, O, diethyl O- p-nitrophenyl phosphorothioate, 20 and 200 μM) to paraoxon at a rate of 23.3–199.3 and 18.7–310.3 pmol/min per mg protein, respectively. p-Nitrophenol, was also formed, at 321.1–769.2 and 406.2–778.3 pmol/min per mg protein. This represented a 16-fold and 2-fold range in capacity to activate and detoxify parathion, respectively. Parathion was activated with an apparent K m of 9–16 μM ( n=3). The activation of parathion (200 μM) was positively correlated with nifedipine oxidation, indicating the involvement of CYP3A. Correlations were not significant with ethoxyresorufin- O-dealkylation (CYP1A1/2), pentoxyresorufin- O-dealkylation (CYP2B6), p-nitrophenol hydroxylation (CYP2E1), paraoxon hydrolysis (A-esterase) or phenylvalerate hydrolysis (B-esterase). Paraoxon formation from parathion was markedly reduced by CYP3A inhibitors. Experiments with EDTA indicated that A-esterase was not functionally important at low levels of paraoxon. Human P450s 3A4 and 3A5 expressed microsomes were the most efficient at biotransforming parathion to paraoxon, although P450s 1A1, 2B6 and 2C8 also catalysed the reaction. This study has determined wide interindividual variations in capacity to metabolise parathion, mainly by CYP3A, which may influence its manifest toxicity.

Comparisons of Phosphorothioate and Phosphate Monoester Transfer Reactions: Activation Parameters, Solvent Effects, and the Effect of Metal Ions

The thermodynamics, pH dependency, solvent effects, and the effect of divalent metal ions have been examined for the hydrolysis reactions of the phosphorothioate monoester p-nitrophenyl phosphorothioate (pNPPT) and compared with those of the corresponding phosphate monoester, p-nitrophenyl phosphate (pNPP). The pH dependency for pNPPT hydrolysis mirrors that for typical phosphate monoesters, with the monoanion being the most reactive species. The ratio of the rate constants for pNPPT hydrolysis to that for pNPP is 1380 for the monoanion reactions and 12.6 for the dianion reactions at 39 °C. The free energy of activation for hydrolysis of the pNPPT dianion in water is 27.9 kcal/mol versus 29.5 kcal/mol for pNPP; for the monoanion reactions the values are 22.2 kcal/mol for pNPPT and 26.8 kcal/mol for pNPP. The free energies of solvation for pNPPT and pNPP are within 0.1 kcal/mol of each other despite the poorer hydrogen bonding ability of sulfur versus oxygen. This minimal difference in ground-state energie...

Possible involvement of beta-adrenergic receptors in the enhancement of nocturnal pineal N-acetyltransferase activity due to parathion administration

The purpose of the present study was to examine the effects of administration of sublethal doses of O, O-diethyl- O- p-nitrophenyl phosphorothioate (parathion) on serum epinephrine (EPI) and norepinephrine (NE), as well as on night-time rat pineal melatonin synthesis, both in the presence and absence of propranolol, a β-adrenergic receptor antagonist. In the first experiment, two groups of adult albino rats were administered parathion orally (1.08 and 2.17 mg/kg/day; the total received by each animal was 6.5 and 13.0 mg/kg body weight over 6 days); another two groups received corn oil only. Animals were killed at 23:00 and 01:00 h by decapitation. Serum EPI was augmented at 01:00 h, but NE was increased at 01:00 and 23:00 h due to administration of the high dose of parathion (13 mg/kg). In the second experiment, two groups of adult male albino rats were administered parathion orally (13 mg/kg); another two groups received an intraperitoneal injection of propranolol (20 mg/kg body weight, 1 h before the lights were turned off). In addition, two groups were given a saline injection. Four hours after darkness onset, pineal N-acetyltransferase (NAT) activity as well as pineal and serum melatonin levels were measured. Parathion by itself significantly augmented nocturnal pineal NAT activity and serum melatonin levels in otherwise untreated rats; the insecticide was ineffective in reference to this enzyme when it was given in conjunction with the β-adrenergic receptor antagonist propranolol. The augmentation of NAT activity by parathion also caused significant reduction in pineal serotonin (5-HT); again, this response was blocked by propranolol treatment. The results are consistent with the idea that parathion influences pineal 5-HT metabolism either at the level of the β-adrenergic receptor or via the sympathetic innervation to the pineal gland.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase by Chlorpyrifos-oxon

Phosphorothionate insecticides such as parathion ( O, O-diethyl O- p-nitrophenyl phosphorothioate) and chlorpyrifos (CPS; O, O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate; Dursban) are metabolically converted by oxidative desulfuration into paraoxon and chlorpyrifos-oxon (CPO). The insecticidal action of chlorpyrifos stems from inhibition of acetylcholinesterase (AChE) by CPO, resulting in severe cholinergic toxicity. Sensory peripheral neuropathy was observed in people exposed environmentally to chlorpyrifos sprayed in confined areas. We have examined the kinetics of inhibition of AChE and butyrylcholinesterase (BChE) by paraoxon and CPO. The bimolecular rate constants ( k i ) for inhibition by paraoxon of recombinant human (rH) AChE, recombinant mouse (rM) AChE, and fetal bovine serum (FBS) AChE were 7.0, 4.0, and 3.2 × 10 5 M −1 min −1. The k i values for the inhibition by CPO of rH AChE, fetal bovine serum AChE, human RBC AChE, Torpedo AChE, and recombinant mouse (rM) AChE were 9.3, 2.2, 3.8, 8.0, and 5.1 × 10 6 M −1 min −1, respectively. Inhibition of human serum BChE, rH BChE, and rM BChE by CPO yielded k i values of 1.65, 1.67, and 0.78 × 10 9 M −1 min −1, respectively. The k i values obtained for BChE from various species were 160- to 750-fold larger than those of AChE from parallel sources. Inhibition of the single-site mutant A 328Y of rH BChE by CPO displayed a 21-fold lower rate than that of wild-type rH BChE ( k i , 7.9 × 10 7 vs 1.67 × 10 9 M −1 min −1). The double mutant of acyl pocket residues of rH AChE, F 295L/F 297V, was inhibited by CPO with a 150-fold larger k i than wild type (1.5 × 10 9 vs 1.0 × 10 7 M −1 min −1). The increased rate obtained with the double mutant displaying characteristics of the BChE active center provides a rationale for higher efficacy of CPO scavenging by BChE, compared with AChE.

Methyl parathion activation by a partially purified rat brain fraction

Organophosphorus pesticides are one of the most commonly used insecticide classes. They act through a potent inhibition of acetylcholinesterase (AChE). Many of them must undergo transformation into the corresponding oxon analogs to inhibit AChE. This study showed that a brain tissue subfraction transformed methyl parathion ( O, O-dimethyl O- p-nitrophenyl phosphorothioate) in vitro. Methyl parathion activation was assayed by solvent extraction of the products followed by HPLC and GC-MS analyses and, indirectly, by the inhibition of AChE present in the incubation mixture. The lack of impairment of AChE after 2 h of incubation of the brain subfraction with methyl parathion and, alternatively, with NADPH, CO, SKF 525-A, piperonyl butoxide or nitrogen indicated that this brain subfraction transformed methyl parathion without the involvement of a mixed-function oxidative pathway.The results from HPLC analysis did not show a peak corresponding to methyl paraoxon ( O, O-dimethyl O- p-nitrophenylphosphate), but showed the production of an unidentified peak which eluted nearby standard methyl parathion (retention times of 10.65 and 8.86 min, respectively). GC-MS analysis suggested that the unidentified product could be a methyl parathion isomer.

Interaction of ethanol and the organophosphorus insecticide parathion

Phosphorothioate insecticides such as parathion ( O, O-diethyl- O- p-nitrophenyl phosphorothioate) undergo P450-dependent oxidative desulfuration, leading to both activation and detoxification of these compounds. Consequently, alterations in P450-dependent oxidative desulfuration may affect the acute toxicities of these insecticides. In the present study, pretreatment of mice with 15% ethanol in the drinking water for 6 days antagonized the acute toxicity of parathion, but not its toxic metabolite paraoxon ( O, O-diethyl- O- p-nitrophenyl phosphate), suggesting that ethanol affected the oxidative desulfuration of this insecticide. The presence of ethanol within hepatic microsomal incubations did not alter the P450-dependent formation of paraoxon (activation) and p-nitrophenol (detoxification), although p-nitrophenol levels were increased in the presence of ethanol as a result of inhibition of its biotransformation to 4-nitrocatechol by CYP2E1. Ethanol exposure reduced hepatic pyruvate levels, but had no effect on levels of lactate, isocitrate, α-ketoglutarate, and malate. Calculation of cytosolic NAD + NADH and cytosolic NADP + NADPH redox ratios did not reveal any detectable difference in redox state between control and ethanol-treated mice. Since ethanol did not alter directly the P450-dependent activation or detoxification of parathion, and did not decrease NADPH levels, ethanol's antagonism of the acute toxicity of parathion may result from reduced availability of O 2.

Inhibition of human fetal brain acetylcholinesterase: Marker effect of neurotoxicity

Human fetal brains were obtained after medical termination of pregnancy at 8-10 wk from informed patients. A definite regionalization of AChE was found in the brain of the fetus, with cerebellum recording the highest and cerebral hemisphere the lowest activity. Optimum conditions for the cerebellar AChE activity were determined with respect to molarity of the buffer, pH, temperature, and concentrations of substrate (acetylthiocholine iodide), activators (NaCl, MgCl2), and thiol indicator (dithiobisnitrobenzoic acid). In vitro inhibition of cerebellar AChE with two commercial pesticides, Metacid-50 (O,O-dimethyl p-nitrophenyl phosphorothioate) and carbaryl (N-methyl naphthyl-1-carbamate), were compared with pure anticholinesterase agents, diisopropylfluorophosphate (DFP) and physostigmine (eserine). In general, organophosphates are more neurotoxic than carbamate compounds, as evidenced by higher degree of AChE inhibition by DFP and Metacid-50 as compared to eserine and carbaryl. Assays were also done with psychotropic drugs by employing the procedure of in vitro AChE inhibition kinetics, and it was found that psychotropic drugs are less potent than organophosphate and carbamate compounds. Results indicate that pure and commercial organophosphates and carbamates and psychotropic drugs are all able to significantly alter the AChE activity. Thus exposure of the mother to these environmental toxicants may adversely affect the fetal neural functions.

In vivo penetration and metabolism of methyl parathion in larvae of the tobacco budworm, Heliothis virescens (F.), fed different host plants

The effect of wild tomato, Lycopersicon hirsutum f. glabratum (accession PI 134417), and peppermint, Mentha piperita L., on the susceptibility, penetration, and metabolism of methyl parathion, O,O-dimethyl O- p-nitrophenyl phosphorothioate, was studied in larvae of the tobacco budworm, Heliothis virescens F. Third-instar larvae fed wild tomato or peppermint leaves for 1 day were 3.3- and 2.7-fold more tolerant to methyl parathion, respectively, as compared to insects fed an artificial diet. These tolerance levels were only 2.2- and 1.7-fold, respectively, in fifth-instar larvae when fed leaves of the same plant species for 1 day. Penetration studies did not indicate any differences in the rate of penetration of methyl parathion in larvae fed different diets. Methyl parathion injected into fifth-instar larvae was converted into three chloroform-soluble and five water-soluble metabolites. Five hours after injection, the extent of methyl parathion metabolism was greater in larvae fed wild tomato leaves (77.4%) or peppermint leaves (72.8%), than in those fed an artificial diet (64.55%). The major metabolite was p-nitrophenol and its formation was higher in larvae fed wild tomato leaves (49.4%) than in larvae fed peppermint leaves (44.23%), which in turn were higher than those fed an artificial diet (38.19%). These data suggested that the two plants induced enzymes responsible for detoxifying methyl parathion.

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments.

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.

Relative persistence of hexachlorocyclohexane, methyl parathion and carbofuran in an alluvial soil under flooded and non-flooded conditions

The relative persistence of the γ-isomer of hexachlorocyclohexane (γ-HCH, organochlorine), methyl parathion ( O- O-dimethyl O- p-nitrophenyl phosphorothioate, organophosphate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate, carbamate) in an alluvial soil was studied under flooded and non-flooded conditions. Under flooded conditions, γ-HCH was the least persistent of the three insecticides followed by methyl parathion and carbofuran, but under non-flooded conditions, γ-HCH was the most stable followed by carbofuran and methyl parathion. A major portion of the 14C in γ-HCH and methyl parathion was not accounted for, especially under flooded conditions, possibly due to the formation of volatile products. Formation of soil-bound residues from γ-HCH and methyl parathion was negligible. However, during metabolism of carbofuran, its hydrolysis product, carbofuran phenol, and bound residues accumulated in the soil in substantial quantities. The data generated from these concurrently incubated studies demonstrate that the relative persistence of insecticides with different functional groups in the soil is governed by the moisture regime.

PH-dependent oxygen exchange of phosphate and hydrolysis (of sulfur) from thiophosphate catalyzed by acid phosphatases

Phosphate oxygen exchange and thiophosphate hydrolysis reactions catalyzed by a number of homogeneous acid phosphatase isoenzymes were studied as a function of pH. Data are presented for the pH dependence of the phosphate oxygen—water oxygen exchange reaction catalyzed by human prostatic acid phosphatase. The K cat for phosphate oxygen exchange is approximately constant from pH 2.7 to 7.0 and declines at high pH. This pH dependence resembles that of V for typical substrate hydrolysis reactions. Human prostatic acid phosphatase is shown to catalyze the hydrolysis of inorganic thiophosphate to phosphate ion. The rate of thiophosphate ion hydrolysis is maximal in the pH range 3 to 5 and decreases at higher pH. Hydrolysis reactions of phosphomonoesters and related phosphorothioatemonoesters were compared using acid phosphatases from animal and plant sources. Wheat germ acid phosphatase catalyzes the hydrolysis of adenosine 5′-phosphorothioate and p-nitrophenyl phosphorothioate, though at a much slower rate than the hydrolysis of the corresponding phosphomonoesters. Similarly, human prostatic acid phosphatase hydrolyses adenosine 5′-phosphorothioate at a very slow rate ( k cat = 2.3 × 10 −2s −1), whereas the corresponding phosphomonoester is hydrolyzed four orders of magnitude more rapidly. A human liver acid phosphatase isoenzyme hydrolyses AMP and p-nitrophenyl phosphate at a rate of 1.0 and 1.2 × 10 2 s −1, respectively, but adenosine 5′-phosphorothioate and p-nitrophenyl phosphorothioate are not detectably hydrolyzed by this enzyme. A low-molecular-weight acid phosphatase from bovine liver hydrolyzed p-nitrophenyl phosphate at a rate of 35 s −1, but it failed to hydrolyze AMP, adenosine 5′-phosphorothioate, or p-nitrophenyl phosphorothioate. Various thermodynamic parameters for phosphate oxygen exchange and for the hydrolysis of thiophosphate and p-nitrophenyl phosphate catalyzed by human prostatic acid phosphatase were also measured. The activation energy for phosphate oxygen exchange catalyzed by human prostatic acid phosphatase is 17 kcal mol −1 while the activation energy for the hydrolysis of p-nitrophenyl phosphate is 10.9 kcal mol −1. K m(app) for the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase decreases from 0.61 m m at 7 °C to 0.10 m m at 37 °C.

Degenerative changes in skeletal muscle of hens with tri- ortho-cresyl phosphate-induced delayed neurotoxicity: Altered acetylcholinesterase molecular forms and increased plasma creatine phosphokinase activity

Degenerative changes in skeletal muscle were studied in hens with a delayed neuropathy induced with TOCP (tri- ortho-cresyl phosphate) that was characterized by axonal fragmentation in the sciatic nerve and by paralysis of the legs. Mean muscle weight and fiber diameter were significantly decreased in the musculus gastrocnemius, a leg muscle innervated by a branch of the sciatic nerve. The muscle fibers also exhibited abnormal morphology and high extrajunctional AChE activity in the muscle fiber sarcoplasm. Biochemical studies showed that the conentrations of acetylcholinesterase (AChE, EC 3.1.1.7) and nonspecific cholinesterase (EC 3.1.1.8) activities were doubled in the gastrocnemius of hens with delayed neurotoxicity compared to control. Sedimentation of the AChE molecular forms on sucrose density gradients showed that the relative activity of the 7 S form more than doubled and that of the 20 S form was decreased by 57% compared to normal hens. Following the onset of delayed neurotoxicity, creatine phosphokinase (EC 2.7.3.2) activity in the plasma was four times normal, and it was decreased by 56% in the gastronemius. These changes were not observed in birds treated with parathion ( O,O-diethyl- p-nitrophenyl phosphorothioate), which suggested that the alterations were associated with the delayed neuropathy. Muscle atrophy occurred in the m. pectoralis major of birds treated with TOCP and parathion, but this finding may have been a secondary effect of both organophosphates.