Role of genetic polymorphism of human plasma paraoxonase/arylesterase in hydrolysis of the insecticide metabolites chlorpyrifos oxon and paraoxon

Plasma paraoxonase is a polymorphic enzyme that hydrolyzes paraoxon, the neurotoxic, active metabolite of the insecticide parathion. This enzyme is specified by at least two alleles with frequencies of about .7 and .3 among Caucasoid populations. A specific assay was developed that measured the activity of human plasma paraoxonase without interference from serum albumin which contributes significantly to the hydrolytic breakdown of paraoxon at the high pH values used in many previous assays. There was an 11-fold variation in paraoxonase activities, and the population distribution was at least bimodal. However, this specific assay did not improve the discrimination between the three genetic classes: (1) homozygotes for the low-activity allele, (2) heterozygotes, and (3) homozygotes for the high-activity allele. Chlorpyrifos oxon--the neurotoxic metabolite of the organophosphorus insecticide chlorpyrifos (Dursban)--was hydrolyzed by the same plasma fraction that hydrolyzed paraoxon. There was only four- to fivefold variability in enzyme activity, and the population distribution was unimodal. Homozygotes for low paraoxonase activity ranged over almost the entire spectrum of chlorpyrifos oxonase activity. Possible differences in susceptibility to chlorpyrifos toxicity therefore are unlikely to be predicted by the paraoxonase genotype alone. The ratio of paraoxonase over that of chlorpyrifos oxonase provided an excellent method for genetic typing of the paraoxonase polymorphism, as did the substitution of phenylacetate for chlorpyrifos as the substrate.     

Seasonal variation of arylesterase activity and its consequence for enzymatic hydrolysis of phenylacetate]

Studies on red cell arylesterase activity were performed for two years by potentiometric titration using phenylacetate as substrate. From April to October enzymes had higher arylesterase activity than the rest of the year. During that period an increase of arylesterase activity by L-phyenyl-alanine had not been shown.     

Two specific and simple methods for genotyping of the paraoxonase/arylesterase A/B polymorphism

We developed an amplification refractory mutation system (ARMS) and mutagenically separated PCR (MS-PCR) assays for genotyping of the paraoxonase/arylesrase (PONA) A/B polymorphism as practical, reliable, and more economic alternatives to the conventional restriction fragment length polymorphism (RFLP) technique for detection of this mutation.     

Identification of residues essential for human paraoxonase (PON1) arylesterase/organophosphatase activities

Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.     

Optimization of the enzymatic hydrolysis and analysis of plasma conjugated γ-CEHC and sulfated long-chain carboxychromanols, metabolites of vitamin E

Natural forms of vitamin E are metabolized by ω-hydroxylation and β-oxidation of the hydrophobic side chain to generate urinary-excreted 2-(β-carboxyethyl)-6-hydroxychroman (CEHC) and CEHC conjugates (sulfate, glucuronide, or glucoside). We recently showed that sulfated long-chain carboxychromanols, the conjugated intermediate β-oxidation products, are formed from tocopherols and tocotrienols in human cells and in rats. CEHC conjugates have been quantified after being converted to its unconjugated counterpart by sulfatase/glucuronidase. Although the enzymatic hydrolysis is critical for appropriate quantification of conjugated CEHC, it is not clear whether brief incubation of the plasma with sulfatases/glucuronidases results in complete deconjugation of conjugated CEHC. Here we show that quantitative hydrolysis of the conjugated vitamin E metabolites in the plasma requires an extraction procedure using methanol/hexane (2 ml/5 ml) and an overnight sulfatase/glucuronidase hydrolysis. Using this procedure, we demonstrate that conjugated γ-CEHC and some sulfated long-chain carboxychromanols are fully deconjugated. In contrast, direct enzymatic hydrolysis of the whole plasma underestimates the conjugated metabolites by at least threefold. This protocol may be also useful for the analysis of other conjugated phenolic compounds in complicated biological matrices such as plasma.     

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.     

Differential modulation of organophosphate-sensitive muscarinic receptors in rat brain by parathion and chlorpyrifos

We previously reported similar levels of brain cholinesterase inhibition but marked differences in toxicity following acute maximum tolerated doses of the organophosphate pesticides parathion and chlorpyrifos. Because extensive acetylcholinesterase inhibition often induces compensatory changes in cholinergic receptor populations, we compared the effects of parathion and chlorpyrifos on brain muscarinic receptors. Adult male rats were treated with vehicle or the maximum tolerated dose of parathion (18 mg/kg, sc) or chlorpyrifos (279 mg/kg, sc) and observed for signs of acute toxicity. Similarly treated animals were sacrificed at 2, 7, or 14 days after treatment for measurement of cholinesterase activity and binding to the nonselective muscarinic antagonist [³H]quinuclidinyl benzilate, the M2-preferential antagonist [³H]AFDX-384, and the high-affinity agonist [³H]cis-methyldioxolane. More acute toxicity was noted after parathion treatment. Both insecticides caused similar levels (> 85%) of maximal cholinesterase inhibition and reductions (up to 55%) in atropine-sensitive quinuclidinyl benzilate binding (i.e., total muscarinic receptors) and [³H]AFDX-384 binding in cortex and striatum. Parathion also reduced, whereas chlorpyrifos increased, total muscarinic receptor binding and [³H]AFDX-384 binding in the cerebellum. When tissues were preincubated with paraoxon (10 μM), radiolabeling of a subset of quinuclidinyl benzilate binding sites was blocked and the apparent densities of these organophosphate-sensitive receptors in all three tissues were decreased (16% maximal) by parathion but increased (up to 37%) by chlorpyrifos. Similarly, parathion decreased whereas chlorpyrifos increased [³H]cis-methyldioxolane binding sites in all three brain regions. We propose that differential modulation of these organophosphate-sensitive muscarinic receptors contributes to differences in acute toxicity following exposure to these pesticides.     

Inhibitory potency against human acetylcholinesterase and enzymatic hydrolysis of fluorogenic nerve agent mimics by human paraoxonase 1 and squid diisopropyl fluorophosphatase

A wide range of toxic organophosphorus pesticides and nerve agents is effectively hydrolyzed by the structurally related phosphotriesterase enzymes paraoxonase (PON1) from human plasma and diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris. Both enzymes have potential use as medical countermeasures and decontaminants. Enhanced enzymatic activity, stereochemical preference, and substrate variety are still the focus of ongoing research. Derivatives of pesticides and nerve agents bearing a fluorogenic leaving group were introduced for high-throughput screening of mutant libraries recently. We report the inhibitory potency of fluorogenic organophosphorus compounds with three different leaving groups [3-chloro-7-oxy-4-methylcoumarin, 7-oxy-4-methylcoumarin, 7-oxy-4-(trifluoromethyl)coumarin] toward human acetylcholinesterase (AChE) and report kinetic data for the enzymatic hydrolysis of these compounds by PON1 and DFPase. This is the first report of the hydrolysis of a substrate bearing a P-O bond to the leaving group by DFPase (its activity was believed to be restricted to cleavage of P-F and P-CN bonds). The reactivity of the enzymes toward the substrates is explained on the basis of structural reasoning and computational docking studies. We demonstrate that fluorogenic organophosphorus compounds can serve as valuable models for enzyme screening but also show that differences and limitations exist and have to be taken into account. The importance of using protein from human sources to obtain toxicological data for potential in vivo use is highlighted.     

Effects of red wine consumption on serum paraoxonase/arylesterase activities and on lipoprotein oxidizability in healthy-men

Although there is a general consensus concerning the lower risk for cardiovascular disease in moderate drinkers, the mechanisms responsible for the cardioprotective effect of red wine remain unknown. It has been proposed that increased serum paraoxonase activity may be a mechanism of action underlying reduced cardiovascular disease risk in moderate drinkers, since paraoxonase inhibits lipoprotein oxidation. The aim of this study was to investigate the effects of red wine consumption on serum paraoxonase/arylesterase activities and on lipoprotein oxidizability in healthy-men. Fourteen healthy-men were included in the study. The subjects consumed 0.375 g alcohol / kg body weight for 3 weeks. Paraoxonase and arylesterase activities were studied spectrophotometrically. Oxidizability of apolipoprotein B-containing lipoproteins were determined, after separating them with precipitation method, by incubating with copper-sulfate. Paraoxonase activity did not change, however arylesterase activity significantly decreased after red wine consumption (P < 0.01). There was a reduced susceptibility of apolipoprotein B-containing lipoproteins to copper-sulfate induced oxidation after red wine consumption (P < 0.01). Our results support that red wine protects lipoproteins against oxidation, however there was not any significant change in serum paraoxonase activity after red wine consumption.KEY WORDS: Red wine; Paraoxonase; Arylesterase; Lipoprotein oxidation     

Selective high-performance liquid chromatographic assays for hydralazine and its metabolites in plasma of man

Selective high-performance liquid chromatographic assays for hydralazine (I), hydralazine pyruvic acid hydrazone (II) and the acetylation metabolites, namely s-triazolo[3,4-a]-phthalazine (V) and 3-hydroxymethyl (VI) and 3-methyl-s-triazolo[3,4-a]phthalazine (VII) in human plasma were developed. Utilizing the fluorescence of these compounds or their derivatives the limits of detection could be extended down to 5 nmole/l (1 ng/ml) for I, 1 nmole/l (0.2 ng/ml) for II and 0.5 nmole/l (0.1 ng/ml) for V–VII. The intra-assay coefficients of variation for the assays ranged from 2 to 7% over the concentration range 5.0 to 0.05 μmole/l and the inter-assay variability in the slope of the standard curves ranged from 4 to 8%. An improved method for measuring the sum of I plus all its hydrazones (apparent I) was also developed. On addition of I to fresh plasma at 37°, half the added I was converted to II within 15 min and there was no detectable level of I, 2 h after the addition. The plasma level—time course of I, and its metabolites in a healthy volunteer (slow acetylator) following separate oral and intravenous administrations of I indicated that I contributed only a small fraction (4.3 and 4.7% respectively) to the area under the plasma level—time curve of apparent hydralazine.     

Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase

Reducing the enzyme loadings for enzymatic saccharification of lignocellulose is required for economically feasible production of biofuels and biochemicals. One strategy is addition of small amounts of synergistic proteins to cellulase mixtures. Synergistic proteins increase the activity of cellulase without causing significant hydrolysis of cellulose. Synergistic proteins exert their activity by inducing structural modifications in cellulose. Recently, synergistic proteins from various biological sources, including bacteria, fungi, and plants, were identified based on genomic data, and their synergistic activities were investigated. Currently, an up-to-date overview of several aspects of synergistic proteins, such as their functions, action mechanisms and synergistic activity, are important for future industrial application. In this review, we summarize the current state of research on four synergistic proteins: carbohydrate-binding modules, plant expansins, expansin-like proteins, and Auxiliary Activity family 9 (formerly GH61) proteins. This review provides critical information to aid in promoting research on the development of efficient and industrially feasible synergistic proteins.     

Spectrophotometric and high-performance chromatographic assays of hydroperoxides by the iodometric technique

Samples containing between 1 and 50 nmol of hydroperoxides in oxygen-free methanol-acetic acid containing I- were incubated at 50 degrees C. This ensured reduction of all hydroperoxides tested in 15 min. Addition of sufficient Cd acetate to combine with the remaining I- allowed reading of absorbance of the I3- produced in open cuvettes. The absorbance was stable for several hours. It had a maximum at 358 nm with molar extinction coefficient of 2.97 X 10(4) 1 mol-1 cm-1. Sensitivity of the assay could be improved by injecting the I3- into a C-18 HPLC column eluted with methanol-water-acetic acid solution. Both methods are potentially suitable for assay of hydroperoxides in a wide range of biological materials.     

Mathematical model for enzymatic hydrolysis and fermentation of cellulose by Trichoderma

This paper describes a mathematical model for the enzymatic hydrolysis and fermentation of cellulose by Trichoderma reesei. The principal features of the model are the assumption of two forms of cellulose (crystalline and amorphous), two sugars (cellobiose and glucose), and two enzymes (cellulase and β-glucosidase). An inducer–repressor–messenger RNA mechanism is used to predict enzyme formation, and pH effects are included. The model consists of 12 ordinary differential equations for 12 state variables and contains 38 parameters. The parameters were estimated from four sets of experimental data by optimization. The results appear satisfactory, and the computer programs permit simulation of a variety of system changes.     

Inhibition of the enzymatic hydrolysis of cellulose by ethanol

Ethanol inhibits the cellulase from Trichoderma reesi progressively and linearly up to 65 g/L. The inhibition of this magnitude presents a potential problem in the simultaneous saccharification and fermentation, presently a norm of the process scheme in ethanol production from biomass.     

Simultaneous degradation of the pesticides methyl parathion and chlorpyrifos by an isolated bacterial consortium from a contaminated site

The simultaneous degradation of the pesticide methyl parathion and chlorpyrifos was tested using a bacterial consortium obtained by selective enrichment from highly contaminated soils in Moravia (Medellin, Colombia). Microorganisms identified in the consortium were Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp. In culture medium enriched with each of the pesticides, the consortium was able to degrade 150 mg l⁻¹ of methyl parathion and chlorpyrifos in 120 h. When a mixture of 150 mg l⁻¹ of both pesticides was used the percentage decreased to 72% for methyl parathion and 39% for chlorpyrifos. With the addition of glucose to the culture medium, the consortium simultaneously degraded 150 mg l⁻¹ of the pesticides in the mixture. 4 treatments were carried out in soil that included the addition of glucose with microorganisms, the addition of sugar cane with microorganisms, microorganisms without nutrient addition and without the addition of any item. In the treatment in which glucose was used, degradation percentages of methyl parathion and chlorpyrifos of 98% and 97% respectively were obtained in 120 h. This treatment also achieved the highest percentage of reduction in toxicity, monitored with Vibrio fischeri.     

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Genetic and biochemical evidence for the lack of significant hydrolysis of soman by a Flavobacterium parathion hydrolase.

Pure recombinant Flavobacterium parathion hydrolase (an organophosphorus acid anhydrase) from Streptomyces lividans was found to hydrolyze the toxic nerve agent soman at only 0.1% of the rate observed with parathion as substrate. Studies with wild-type and recombinant strains of S. lividans support the lack of significant soman breakdown by the hydrolase and also indicate the presence in S. lividans of other significant hydrolytic enzymatic activity towards soman.     

Genetic and biochemical evidence for the lack of significant hydrolysis of soman by a Flavobacterium parathion hydrolase.

Pure recombinant Flavobacterium parathion hydrolase (an organophosphorus acid anhydrase) from Streptomyces lividans was found to hydrolyze the toxic nerve agent soman at only 0.1% of the rate observed with parathion as substrate. Studies with wild-type and recombinant strains of S. lividans support the lack of significant soman breakdown by the hydrolase and also indicate the presence in S. lividans of other significant hydrolytic enzymatic activity towards soman.