Ion selective electrode for 2,4-dichlorophenoxyacetic acid

An electrode responsive to 2,4-D (2,4-dichlorophenoxyacetic acid) was constructed by dissolving tetrazolium derivatives as an ion exchanger in a liquid membrane. The electrode exhibited rapid and Nernstian response to solutions of 2,4-D over the concentration range 10(-1) to 10(-4)M. The presence of diverse substances such as acetate, benzoate, and 3-indoleacetate showed no appreciable effect on the electromotive force of the electrode.     

Interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) with cell and model membranes

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a component of the "agent orange' whose toxicity has been extensively studied without definite conclusions. In order to evaluate its perturbing effect upon cell membranes, 2,4-D was made to interact with human erythrocytes and molecular models. These studies were performed by scanning electron microscopy on red cells, fluorescence spectroscopy on dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles and X-ray diffraction on multilayers of DMPC and dimyristoylphosphatidylethanolamine (DMPE). It was observed that 2,4-D induced a pronounced shape change to the erythrocytes. This effect is explained by the herbicide interaction with the outer monolayer of the red cell membrane.     

Development of an amperometric flow injection immunoanalysis system for the determination of the herbicide 2,4-dichlorophenoxyacetic acid in water

An amperometric flow injection immunoanalysis (FIIA) system based on an immunoreactor with immobilized biocomponents on a silica surface has been developed for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). In the antigen coating mode the hapten was immobilized and monoclonal primary antibody against 2,4-D together with alkaline phosphatase (AP)-labelled secondary antibody were used as sensing elements in a titration assay. In the antibody coating mode a biotinylated monoclonal antibody was immobilized on the surface of the immunoreactor and a 2,4-D-AP-conjugate was used for detection. For electrochemical measurements p-aminophenol enzymatically generated from p-aminophenyl phosphate was oxidized at a carbon working electrode at +150 mV versus Ag/AgCl. The system enabled the determination of 2,4-D in drinking water samples in the range from 0.2 to 70 micrograms/l. The whole system was computer controlled with a measuring time of 12 min for one determination.     

Taurine conjugation of 2,4-dichlorophenoxyacetic acid and phenylacetic acid in two marine species

1. At least 50% of a dose of 14C-labelled 2,4-dichlorophenoxyacetic acid or phenylacetic acid was excreted in urine in 48 hours after administration to dogfish shark or flounder. 2. For both compounds, more than 90% of the urinary 14C was present as a single metabolite. 3. Each metabolite was the taurine conjugate of the administered compound.     

Mechanism mediating basolateral transport of 2,4-dichlorophenoxyacetic acid in rat kidney

The organic anions, p-aminohippurate (PAH) and fluorescein, are transported across the basolateral membrane of the renal proximal tubule in exchange for intracellular alpha-ketoglutarate (alpha KG), a mechanism indirectly coupled to sodium via Na+/alpha KG cotransport. To determine whether this mechanism mediates the basolateral transport of other organic anions, transport of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in rat renal cortical slices and basolateral membrane vesicles. In slices, uptake of 2,4-D increased steadily over time, approaching steady-state tissue/medium ratios of approximately 8 after 60 min. Probenecid, PAH and chlorophenol red inhibited steady-state uptake of 2,4-D. Accumulation of 10 microM 2,4-D was stimulated 2-fold by 60 microM glutarate; other dicarboxylic acids failed to stimulate uptake. In the presence of sodium, the addition of 5 mM LiCl or 2 mM ouabain to the bathing medium abolished glutarate stimulation. Removal of sodium from the bathing medium reversibly inhibited uptake as much as 75%. Furthermore, PAH inhibited 2,4-D uptake by slices in a dose-dependent manner, and increasing the external 2,4-D concentration decreased the inhibitory potency of PAH. In basolateral membrane vesicles, unlabeled 2,4-D inhibited sodium glutarate-coupled uptake of 3H-labeled PAH and 2,4-D in a concentration-dependent manner. Moreover, concentrative uptake of 2,4-D into vesicles could be driven by an outwardly directed gradient of glutarate or alpha KG that was generated by lithium-sensitive Na+/dicarboxylate cotransport or imposed experimentally. An outwardly directed gradient of unlabeled 2,4-D or PAH also stimulated uptake of 2,4-D. Based on these data, basolateral accumulation of 2,4-D by the renal proximal tubule is mediated by 2,4-D/alpha KG exchange, a mechanism energetically coupled to Na+/alpha KG cotransport and shared with PAH.     

Circadian periodic response of Phaseolus vulgaris l. to 2,4-dichlorophenoxyacetic acid

The susceptibility of bean plants, Phaseolus vulgaris L., cv. Executive, to 2,4-dichlorophentoxyacetic acid (2,4-D) appeared to depend upon the time of application. Oscillations in response to 2,4-D were evident in plants subjected to conditions of alternating light and dark spans, continuous illumination or darkness. The fresh and dry weight of plant material was generally less when 2,4-D was applied to plants near the later portions of the light span.     

Altered behavioral responses in 2,4-dichlorophenoxyacetic acid treated and amphetamine challenged rats

Although the mechanism of 2,4-D neurotoxicity remains unknown the serotonergic system appears to mediate some of the effects of 2,4-D in rats as reported in our previous studies. In the present study we examine the concept that a challenge to a system may overcome compensatory mechanisms and thereby reveal otherwise hidden neurotoxicant-induced damage. We report the behavioral results of 50 or 100 mg/kg 2,4-Dichlorophenoxyacetic acid (2,4-D) acute exposure plus amphetamine challenge of rats. The "Serotonin Syndrome" (SS) involving prominently head movement, piloerection, moist fur, backing, hunching, Straub tail, fore paw tapping of the nose and pivoting, was exhibited by these rats, being females more affected than males. Immobility, social apathy and asymmetry were also observed. All behaviors were not seen in the 2,4-D treated rats. Stereotyped behaviors were observed earlier and were more prolonged in 2,4-D treated and amphetamine challenged rats than in rats treated only with 5 or 10 mg/kg amphetamine. Spiperone blocked all the SS behaviors. In addition, in these rats, rearing and rotation behaviors were showed and were also sex dependent. We also demonstrate that haloperidol, in a non cataleptic dose, induced catelepsy in 2,4-D treated rats. 2,4-D appears to act through serotonergic and dopaminergic mechanisms. The intensity of the response is sex dependent. Our study demonstrates that 2,4-D plus amphetamine induces a Serotonergic Syndrome plus additional Dopaminergic modulation.     

Ascorbic acid-dependent turnover and reactivation of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase using thiophenoxyacetic acid

The first step in catabolism of the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is catalyzed by 2,4-D/alpha-ketoglutarate (alpha-KG)-dioxygenase (TfdA) in Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. This oxygen- and ferrous-ion-dependent enzyme couples the oxidative decarboxylation of alpha-KG (yielding CO2 and succinate) with the oxidation of 2,4-D to produce 2,4-dichlorophenol and glyoxylate. TfdA was shown to utilize thiophenoxyacetic acid (TPAA) to produce thiophenol, allowing the development of a continuous spectrophotometric assay for the enzyme using the thiol-reactive reagent 4,4'-dithiodipyridine. In contrast to the reaction with 2,4-D, however, the kinetics of TPAA oxidation were nonlinear and ascorbic acid was found to be required for and consumed during TPAA oxidation. The ascorbic acid was needed to reduce a reversibly oxidized inactive state that was formed by reaction of the ferrous enzyme with oxygen, either in the absence of substrate or in the presence of TPAA. The dependency on this reductant was not due to an uncoupling of alpha-KG decarboxylation from substrate hydroxylation, as has been reported for several other alpha-KG-dependent hydroxylases. Significantly, the rate of formation of this reversibly oxidized species was much lower when the enzyme was turning over 2,4-D. Evidence also was obtained for the generation of an inactive enzyme species that could not be reversed by ascorbate. The latter species, not associated with protein fragmentation, arose from an oxidative reaction that is likely to involve hydroxyl radical reactions. On the basis of initial rate studies, the kcat and Km values for TPAA were estimated to be 20-fold lower and 80-fold higher than the corresponding values for 2,4-D. The results are incorporated into a model of TfdA reactivity involving both catalytic and inactivating events.     

Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and mechanism of the interaction between human serum albumin and monomeric haemin

Previous studies showed hyperre-sponsiveness to human growth hormone (hGH) in men with myotonic or limb girdle dystrophies (MMD or LGD). Because polyamines may mediate some actions of hGH, we have now investigated polyamine metabolism in these and other dystrophies. Under metabolic balance study conditions, serum and urine levels of putrescine (Pu), spermidine (Sd), spermine (Sm), and cadaverine (Cd) were measured in six normal men (36-44 yr), four men with MMD (38-44 yr), and three men with LGD (30-36 yr), before and during treatment with 0.532 U/kg body wt ((3/4)/d) of hGH. Daily balances of N, P, and K were also monitored. In the normal subjects, hGH did not influence elemental balances or serum and urine polyamines. In MMD, hGH caused significant retention of N, P, and K (P < 0.005). Basal levels of Sm and Cd were significantly elevated above normal (P < 0.005), and Pu, Sm, and Cd increased two- to fourfold above basal during hGH treatment (P < 0.005). In LGD, hGH also caused retention of N, P, and K. Basal levels of nearly all the polyamines (not serum Pu) were significantly above normal in serum and urine (P < 0.05). During hGH treatment, all four polyamines rose significantly above basal (P < 0.005). Serum and urine polyamine levels in five boys with Duchenne muscular dystrophy, age 8-13, did not differ from those in five age-matched normal boys. Skeletal muscle polyamines were measured in five men (31-40 yr) without muscle disease and in three men with LGD (30-38 yr). Average concentrations of Pu, Sd, Sm, and Cd were 46, 306, 548, and 61 nmol/g wet wt in LGD and 1, 121, 245, and 14 in the normal subjects, respectively (P < 0.05 in each instance). Polyamines were determined in skeletal muscle, liver, kidney, and brain of male mice with hereditary muscular dystrophy and in age- and sex-matched normal controls. Pu, Sd, Sm, and Cd levels were two to three times higher than normal in muscle, but did not differ in liver, kidney, and brain. Similar findings were made in male hamsters with hereditary dystrophy and in their controls. The abnormality in hamster muscle polyamines appeared between 1 and 6 wk of age and persisted or intensified until 30 wk. These data reveal abnormalities of polyamine metabolism in men with MMD, in men with LGD, and in mice or hamsters with hereditary muscular dystrophy. The polyamine disorder could be related to dystrophic patients' hyperresponsiveness to hGH.     

Metabolism of 2,4-dichlorophenoxyacetic acid. IX. Gas-liquid chromatography of methyl esters of amino acid conjugates

Cyclophosphamide, an immunosuppressive agent, was administered as an additional mode of therapy to 30 patients with idiopathic thrombocytopenic purpura (ITP) refractory to conventional management. Of 22 previously tested by splenectomy an excellent response was achieved in 12, who remained in complete hematologic remission for 14-96 months after therapy was discontinued; a fair response in 3, with definite increase in platelets, but not to normal levels; and a poor response in 7 who failed to improve. Of 8 nonsplenectomized patients who failed to respond to steroids or maintain a response after steroids were discontinued, 4 were considered excellent, 1 required continued therapy to remain in remission (good response), 2 were fair, and 1 was poor. Remission was observed in 2-10 weeks in both groups and appeared to be related to duration of disease; presence of disease for less than 1 year was associated with a much better response to treatment (11 of 15) when compared with disorders lasting over 2 years (6 of 15). Cyclophosphamide therapy offers additional means of treating patients with ITP who fail to respond to conventional therapy and may serve as an alternative to splenectomy when surgery is contraindicated.     

Construction of a physiologically based pharmacokinetic model for 2,4-dichlorophenoxyacetic acid dosimetry in the developing rabbit brain

A physiologically based pharmacokinetic (PBPK) model that describes the kinetics of organic anions by using 2,4-dichlorophenoxyacetic (2,4-D) as a representative compound was constructed for the developing rabbit brain at near-term pregnancy (Gestation Day 30). The model consisted of brain, body, and venous and arterial compartments for the mother which were linked to the fetus by a placenta. Maternal brain compartments in the model were brain plasma, cerebrospinal fluid (CSF), and brain tissue including hypothalamus, caudate nucleus, hippocampus, forebrain, brainstem, and cerebellum. The fetus consisted of brain, body, amniotic fluid, and venous and arterial compartments. the maternal body had both a central and a deep compartment; the fetal body had only one compartment. Maternal blood flow to the fetus was modeled as blood flowing to the placenta, where it was equilibrated before it reached the fetus. The brain uptake was membrane-limited by the blood-brain barrier, with saturable clearance from the CSF into the venous blood by the choroid plexus in both fetus and mother. The model was used to compare concentrations of 2,4-D in maternal and fetal brain, maternal and fetal plasma, and amniotic fluid over time with experimental data from pregnant rabbits given 2,4-D intravenously (1, 10, or 40 mg/kg). The model adequately simulated the 2-hr time course of 2,4-D concentrations in both mother and fetus. With continued development, this generic PBPK model should be a useful tool for evaluating the safety of organic acid neurotoxicants in the developing brain.     

稀有金属离子与巯嘌呤血清白蛋白相互作用的研究

用紫外光谱、荧光光谱研究了多种稀有金属离子与抗癌药物巯嘌呤（mercaptopurine,MP)、血清白蛋白的相互作用;并通过对它们之间的结合常数、结合位置数、作用力类型的研究,为稀有金属离子参与癌症治疗、抗癌药物的合成提供了有益的参考。     

Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites

A lexical decision experiment investigated hemisphere asymmetries in resolving lexical ambiguity within a sentence context. Sentences that biased a single meaning (either dominant or subordinate) of sentence-final ambiguous words were followed by a lateralized target related to the sentence-congruent or -incongruent meaning of the ambiguous word, or an unrelated word. In the RVF sentence-congruent targets were facilitated, while incongruent targets were not primed. In contrast, related targets were facilitated in the LVF, regardless of sentence context. This suggests that selecting the contextually appropriate word meaning requires the left hemisphere, and supports a right hemisphere role in maintaining alternate word senses.     

Reversible binding of ethacrynic acid to human serum albumin: difference circular dichroism study

Intimal proliferation at the interface between prosthetic material and tissue is an intrinsic phenomenon of stenting and the major cause of insufficiency of the transjugular intrahepatic portosystemic shunt (TIPS). For its prevention, a randomized study was performed comparing standard heparin treatment with a combination of trapidil, a drug with anti-platelet-derived growth factor (PDGF) activity, and ticlopidine, a platelet aggregation inhibitor. Ninety patients with cirrhosis who received a transjugular shunt were randomized, and 84 patients completed the trial. Group 1 (n = 42) received a bolus of heparin (12 to 24 U/kg) at shunt placement, followed by 1 week of intravenous and 4 weeks of subcutaneous heparin treatment. Group 2 (n = 42) received the same heparin bolus, followed by a 1-day intravenous heparin treatment and a 6-month treatment with trapidil (400 mg/d) and ticlopidine (250 mg/d). Shunt function was assessed by duplex-sonography and angiography. Stenoses were classified according to their location as type 1 (within the stent) and type 2 (in the draining hepatic vein). The estimated rate of overall stenoses (intention-to-treat analysis) at 1 year showed a significant reduction in patients receiving trapidil and ticlopidine (group 2) as compared with heparin (33 vs. 57%; P =.047). There was no difference in the estimated 1-year rate of type 1 stenoses between the two groups, but there was a significant reduction in type 2 stenoses (group 1: 58%, group 2: 19%; P =.016). The treatment effect continued after withdrawal of the drugs and was accompanied by a decreased incidence of rebleeding. The study demonstrates that the incidence of type 2 stenosis of the transjugular shunt can be reduced by combined inhibition of platelet aggregation and PDGF activity. The findings may be of relevance not only for the transjugular shunt, but also for other stent applications, e.g., vascular and biliary, as well as for bypass and shunt surgery.     

Degradation of atrazine and 2,4-dichlorophenoxyacetic acid by mycorrhizal fungi at three nitrogen concentrations in vitro

Nine mycorrhizal fungi and free-living saprophytic microorganisms were tested for their ability to degrade two chlorinated aromatic herbicides at two herbicide concentrations and three nitrogen concentrations. Radiolabelled 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) were used as substrates at concentrations of 1 and 4 mM. After 8 weeks, none of the cultures tested grew at 4 mM 2,4-D. However, when the 2,4-D concentration was reduced to 1 mM, Phanerochaete chrysosporium 1767 had the highest level of 2,4-D mineralization and degradation under all nitrogen conditions. All cultures tested grew at both atrazine concentrations. In all cases, the ericoid mycorrhizal fungus Hymenoscyphus ericae 1318 had the highest level of atrazine carbon incorporated into its tissue. In general, as the nitrogen concentration increased, the total herbicide degradation increased. All of the cultures, except for Rhizopogon vinicolor 7534 and Sclerogaster pacificus 9011, showed increased degradation at 4 mM compared with 1 mM atrazine. The ability to degrade these two herbicides thus appeared to be dependent on the fungus and the herbicide, with no correlation to fungal ecotype (mycorrhizal versus free living).     

The distribution of fatty acid ethyl esters among lipoproteins and albumin in human serum

Fatty acid ethyl esters (FAEEs) are nonoxidative products of ethanol metabolism and have been implicated as mediators of ethanol-induced organ damage. Previous studies have demonstrated that FAEEs bind to lipoproteins and albumin in human plasma after ethanol ingestion. Analysis of human serum with varying blood ethanol levels and endogenously formed FAEEs revealed a positive correlation between serum FAEE concentration and the percentage of FAEEs associated with lipoproteins, predominantly very low density and low density lipoprotein. Similar results were obtained when increasing amounts of FAEEs were added to serum with zero blood ethanol. Additional studies indicated that free fatty acids and FAEEs do not compete for binding to albumin or lipoproteins. Data support the conclusion that the distribution of FAEEs among their carriers in the serum is dependent on serum FAEE concentration.     

Kinetics of peroxynitrite reaction with amino acids and human serum albumin

An initial rate approach was used to study the reaction of peroxynitrite with human serum albumin (HSA) through stopped-flow spectrophotometry. At pH 7.4 and 37 degreesC, the second order rate constant for peroxynitrite reaction with HSA was 9.7 +/- 1.1 x 10(3) M-1 s-1. The rate constants for sulfhydryl-blocked HSA and for the single sulfhydryl were 5.9 +/- 0.3 and 3.8 +/- 0.8 x 10(3) M-1 s-1, respectively. The corresponding values for bovine serum albumin were also determined. The reactivity of sulfhydryl-blocked HSA increased at acidic pH, whereas plots of the rate constant with the sulfhydryl versus pH were bell-shaped. The kinetics of peroxynitrite reaction with all free L-amino acids were determined under pseudo-first order conditions. The most reactive amino acids were cysteine, methionine, and tryptophan. Histidine, leucine, and phenylalanine (and by extension tyrosine) did not affect peroxynitrite decay rate, whereas for the remaining amino acids plots of kobs versus concentration were hyperbolic. The sum of the contributions of the constituent amino acids of the protein to HSA reactivity was comparable to the experimentally determined rate constant, where cysteine and methionine (seven residues in 585) accounted for an estimated 65% of the reactivity. Nitration of aromatic amino acids occurred in HSA following peroxynitrite reaction, with nitration of sulfhydryl-blocked HSA 2-fold higher than native HSA. Carbon dioxide accelerated peroxynitrite decomposition, enhanced aromatic amino acid nitration, and partially inhibited sulfhydryl oxidation of HSA. Nitration in the presence of carbon dioxide increased when the sulfhydryl was blocked. Thus, cysteine 34 was a preferential target of peroxynitrite both in the presence and in the absence of carbon dioxide.