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.     

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.     

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.     

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)     

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.     

Evidence of a strong interaction of 2,4-dichlorophenoxyacetic acid herbicide with human serum albumin

The interaction of 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) with human serum albumin (HSA) was studied using fluorescence and differential scanning calorimetry (DSC). Fluorescence displacement of 1-anilino-8-naphtalenesulfonate (ANS) bound to HSA was used to evaluate the binding affinity of 2,4-D to HSA. The binding is associated to a high affinity site of HSA located in the IIIA subdomain. The association constant (Kass) of the herbicide was about 5 microM(-1), several times higher than the affinity found for pharmaceutical compounds. This relatively strong interaction with HSA was evidenced by the increase in HSA protein thermostability induced as consequence of herbicide interaction. 2,4-D induces an increase in the midpoint of thermal denaturation temperature from 60.1 degrees C in herbicide free solution to 75.6 degrees C in full ligand saturating condition. The calorimetric enthalpy and the excess heat capacity also increased upon 2,4-D binding. To investigate the possibility of other/s system/s of 2,4-D transport in blood, besides of HSA, the interaction of the herbicide with lipid monolayers was explored. No interaction was detected with any of the lipids tested. The overall results provided evidence that high affinity 2,4-D-HSA complex exhibits enhanced thermal stability and that HSA is the unique transport system for 2,4-D in blood.     

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.     

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).     

Expression of ornithine decarboxylase is transiently increased by pollination, 2,4-dichlorophenoxyacetic acid, and gibberellic acid in tomato ovaries

A cDNA encoding for a functional ornithine decarboxylase has been isolated from a cDNA library of carpels of tomato (Lycopersicon esculentum Mill.). Ornithine decarboxylase in tomato is represented by a single-copy gene that we show to be up-regulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid.     

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.     

Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems

Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.     

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.     

Differential effects of 1-naphthaleneacetic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid on the gravitropic response of roots in an auxin-resistant mutant of arabidopsis, aux1

BACKGROUND/AIMS: Gallbladder mucus itself has been recognized to play an important role in gallstone development. Despite the diverse mechanisms of stone induction and the differences in stone composition, there is a quantitative increase in the epithelial mucus production period before stone formation. As brown pigment stones are found frequently in gallstone disease, we conducted a study on gallbladders with brown pigment stones or combination stones with a brown periphery to evaluate the mucin content in the gallbladder epithelium in comparison to gallbladders with cholesterol stones and those without stones. METHODS: Gallbladder specimens were fixed in 10% formalin immediately after cholecystectomy and then embedded in paraffin. The specimens were sectioned for periodic acid-Schiff-alcian blue (PAS-AB, pH 2.5) double stain to evaluate the intra-epithelial mucin content. The PAS-AB index was calculated as a proportion of the PAS-AB-positive mucin area to the total epithelial area, using a computerized image analyzer. RESULTS: Evaluation of the PAS-AB index on the lining epithelia of gallbladders showed that it was 32.43 +/- 9.96% in gallbladders with brown stones, which is significantly (p < 0.001) higher than in gallbladders with cholesterol stones (15.63 +/- 6. 75%) and gallbladders without stones (9.55 +/- 4.77%). CONCLUSION: The results show that gallbladders with brown stones contain more abundant mucin than gallbladders with cholesterol stones or those without stones. They also suggest that the gallbladder epithelium per se might play a more important role in stone formation in those with brown stones than in those with cholesterol stones.     

Inactivation of alpha-ketoglutarate dehydrogenase during oxidative decarboxylation of alpha-ketoadipic acid

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.     

Ascorbic acid and dehydroascorbic acid as biomarkers of oxidative stress caused by smoking

Using a reliable, newly developed assay for ascorbic acid (reduced form) and dehydroascorbic acid (DHAA; the oxidized form) in plasma, we studied the influence of age, sex, and smoking on 219 healthy, age-stratified, and randomly selected subjects representing the Danish population. The mean (+/-SD) plasma total ascorbic acid (ascorbic acid + DHAA) concentration was lower in smokers (62.8 +/- 24.9 mumol/L) than in nonsmokers (74.9 +/- 23.6 mumol/L) (P < 0.001) and the DHAA content was 1.8 +/- 4.0% of the total ascorbic acid in smokers compared with 0.1 +/- 3.1% in nonsmokers (P < 0.001). A significant inverse correlation between the DHAA fraction and the total ascorbic acid concentration was found in smokers (P < 0.002) but not in smokers; the slopes of the linear regressions were significantly different in the two groups (P < 0.005). The mean plasma concentration of total ascorbic acid was higher in females than in males (P < 0.005); this difference persisted in multivariate analysis when smoking was adjusted for. No age dependence could be identified. The data show that smoking results in severe oxidative stress, depletion of the ascorbic acid pool, and insufficient reduction capacity to maintain ascorbic acid in the reduced form in plasma. We suggest that the additional analysis of DHAA allows further differentiation in the assessment of oxidative stress and may provide an objective way of determining vitamin C requirements in smokers. Preliminary findings suggest that a vitamin C dose that results in a plasma concentration of approximately 70 mumol/L or higher is required in smokers.     

Kinetics of refolding and reactivation of rabbit-muscle aldolase after acid dissociation

Tetrameric rabbit muscle aldolase has been dissociated to the monomer at pH 2.3 and fully reassociated and reactivated at pH 7.6 Kinetics of reactivation and refolding were followed by slow and fast kinetic techniques after neutralization of the acidic enzyme solutions by dilution or rapid mixing. Rate constants (k), reaction orders (n), and activation evergies (E) were calculated from measurements on the time, concentration, and temperature dependence of the reactions. The experimental results prove reactivation at high enzyme concentration (c greater than 4mug/ml) to obey first-order kinetics; at lower concentrations a transition to a higher reaction order is observed. Because of concentration-dependent deactivation at low enzyme concentrations reactivation measurements could not be extended below c approximately o.5 mug/ml. In the accessible concentration range incomplete separation of reassociation and transconformation processes as well as intrinsic residual activity of the isolated subunits lead to an average value of n = 1.40 +/- 0.18. Renaturation as monitored by protein fluorescnce is a multi-step process composed of a fast increase in flurescence emission (first-order rate constant k approximately 15 s-1) and a slow concentration-dependent decrease which parallels the recovery of enzyme activity (n = 1.46 +/- 0.12). The activation energy of both processes is of the order of E = 12-16 Kcal/mol (50-67 kJ/mol). Reassociation is a prerequisite of full catalytic function and native fluorescence.     

Ascorbic acid and reducing agents regulate the fates and functions of S-nitrosothiols

The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL. Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.     

Ascorbic acid flux across mucosal border of guinea pig and human ileum

The unidirectional influx of L-[14C]ascorbic acid (vitamin C) across the mucosal border of guinea pig and human ileum was determined. Influx follows saturation kinetics, indicating that a carrier mechanism is operative. The maximal influx in guinea pig ileum bathed in Ringer is 140 nmol/cm2-h and the ascorbic acid concentration greater than 6 mM , or when the tissue is bathed in Na-free media, influx is approximately linearly related to the ascorbic acid concentration, and absorption by simple diffusion may predominate. With mucosal ascorbic acid concentration of 0.28 mM, influx is reduced by at least 70% when Na-free media is used; however, influx is not dependent on the intracellular Na concentration. The brush border mechanism appears to be stereospecific and not closely coupled to cellular metabolism. A model of transport is favored that features a carrier-mediated transport mechanism for simultaneous entry of ascorbic acid and Na across the brush border. This model is similar in nature to, but functionally distinct from, the sodium-gradient mechanism postulated to effect sugar and amino acid transport in mammalian ileal mucosa.