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.     

Effect of radical scavengers on the inactivation of papain by ascorbic acid in the presence of cupric ions

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.     

Immunohistochemical localisation of the 25 kDa heat shock protein in unstressed rats: possible functional implications

The kinetic properties of ascorbic acid oxidation by light-activated melanin granules demonstrate the presence of a specific reactive site on the melanin granule saturable by ascorbic acid. Increased light intensity increased the Vmax and reduced the Km of this reaction, indicating increased affinity of the active site for ascorbic acid. The kinetics of this reaction are not markedly changed in a reduced-oxygen environment. Ascorbic acid oxidation is competitively inhibited by isoascorbic acid, an epimer of ascorbic acid, while other tested reducing agents are inactive. The Ki for isoascorbic acid is 1 mM, about the same as the Km of ascorbic acid.     

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.     

Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol

A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.     

Autoxidation in milk rich in linoleic acid. II. Modification of the initiation system and control of oxidation

Factors contributing to the initiation of lipid oxidation in cow's and mare's milk containing high levels of polyunsaturated fatty acids were studied. Addition of H2O2 just after milking, in slight excess of the stoichiometric amounts required to destroy ascorbic acid, delayed the development of oxidized flavours in cow's milk high in linoleic acid. Hydrogen peroxide treatment followed by the addition of alpha-or gamma-tocopherols prevented lipid oxidation in cow's milk even when 0.1 mg Cu/l milk was added. When used separately in the presence of Cu these treatments were ineffective as was butylated hydroxyanisole treatment. The lipid and ascorbic acid in mare's milk were remarkably stable to oxidation. Addition of 0.05 or 0.1 mg Cu/l, ethylenediamine tetraacetic acid, neocuproine, or H2O2 had very little effect on the loss of ascorbic acid and lipid oxidation in mare's milk.     

Detection of PCB adducts by the 32P-postlabeling technique

The purpose of this study was to determine whether lower chlorinated biphenyls would be bioactivated to electrophilic metabolites by microsomes alone or in combination with peroxidase. Monochloro- and dichlorobiphenyls were incubated with liver microsomes of rats treated with phenobarbital and beta-naphthoflavone, an NADPH-regenerating system, and deoxyguanosine 3'-monophosphate (dGp). The resultant adducts were analyzed by 32P-postlabeling either following microsomal incubation alone ("preoxidized") or coupled with subsequent oxidation with horseradish peroxidase/H2O ("oxidized"). The incubation of 4-monochlorobiphenyl (4-MCB) resulted in the formation of two minor adducts by microsomal activation alone. However, the oxidized sample showed two additional major adducts. Formation of the latter adducts was almost completely (> 80%) inhibited when the oxidation reaction was performed in the presence of ascorbic acid. The other test mono- and dichlorobiphenyls also formed 1-3 major adducts. Compared with microsomal activation alone, these adducts were enhanced after the oxidation reaction or detected only in the oxidized samples. These data suggest that (1) some adducts of the lower chlorinated biphenyls are derived from arene oxides and (2) many adducts may be formed by metabolism of the parent compounds to catechol and p-hydroquinone species, which are oxidized to semiquinones and/or quinones. The involvement of quinones and/or semiquinones was supported by UV/vis spectroscopic measurements, which showed that metabolites of 4-MCB can be oxidized to products with spectra characteristic of quinones. These data raise the possibility that lower chlorinated biphenyls may be genotoxic and may explain the fact that commercial polychlorinated biphenyl mixtures are complete rodent carcinogens.     

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.     

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.     

2,4-Dichlorophenoxyacetic acid action on in vitro protein synthesis and its relation to polyamines

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the in vitro synthesis of proteins was studied in Chinese hamster ovary cells. A remarkable inhibition of the synthesis of proteins was observed when cells grew for 24 h in presence of 1 mM 2,4-D. This effect was reversed by adding 0.1 mM of the three polyamines (putrescine, spermidine and spermine) to the cultured cells. The mRNA is not altered, indicating that the 2,4-D action is located at a different locus, which may be the ribosomes. From these studies, one can speculate that the alterations in the protein synthesis may be a consequence of the effect of 2,4-D on the polyamine metabolism.     

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.     

The reaction of oxygen with radicals from oxidation of tryptophan and indole-3-acetic acid

The oxidation of tryptophan and indole-3-acetic acid (IAA) by the dibromine radical anion or peroxidase from horseradish in aqueous solution was investigated and compared, especially with respect to the involvement of oxygen and superoxide. Using EPR with spin-trapping, the tryptophanyl radical, generated by either method was found to react with oxygen, although this reaction is too slow to be observed by pulse radiolysis (k < 5 x 10(6) dm3 mol-1 s-1). No superoxide results from this reaction, thus excluding an electron-transfer mechanism and suggesting the formation of a tryptophan peroxyl radical, possibly in a reversible process. These observations imply that in proteins where the tryptophanyl radical exists as a stable species it must either have its reactivity modified by the protein environment or be inaccessible to oxygen. The related molecule LAA is oxidized by either peroxidase or Br2.- to a radical cation that decarboxylates to yield a skatolyl radical. The latter reacts with oxygen to give a peroxyl radical that does not release superoxide. However, O2.- is formed during the peroxidase-catalyzed oxidation of indoleacetic acid. This supports the hypothesis that the peroxidase can act in an oxidase cycle involving ferrous enzyme and compound III, with superoxide as a product.     

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.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

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.     

Desferrioxamine improves burst-forming unit-erythroid (BFU-E) proliferation in haemodialysis patients

Biotransformations with recombinant Escherichia coli expressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISP alpha) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISP beta) was shown to play no role in determining the specificities of these dioxygenases.     

Metabolic alterations in hepatocytes promoted by the herbicides paraquat, dinoseb and 2,4-D

The cytotoxic effects of the herbicides paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) on freshly isolated rat hepatocytes were investigated. Paraquat and 2,4-D (1-10 mM) caused a dose and time dependent cell death accompanied by depletion of intracellular glutathione (GSH) and mirroring increase of oxidized glutathione (GSSG). Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000 fold lower than paraquat and 2,4-D), exhibited moderate effects upon the level of GSH and GSSG. These limited effects are at variance with significant effects upon the adenine and pyridine nucleotide contents. ATP and NADH levels are rapidly depleted by herbicide metabolism. This depletion is observed in the millimolar range for paraquat and 2,4-D and in the micromolar range for dinoseb. 2,4-D completely depletes cellular ATP, with subsequent cell death, as detected by LDH leakage. Paraquat rapidly depletes NADH, according to the redox cycling of the herbicide metabolism. The most effective compound is dinoseb since it exerts similar effects as described for paraquat and 2,4-D at concentrations 1000 fold lower. Simultaneously with NADH and ATP depletion, the levels of ADP, AMP and NAD+ increase in hepatocytes incubated in the presence of the herbicides. In contrast to NADH, the time course and extent of ATP depletion and fall in energy charge correlate reasonably with the time of onset and rate of cell death. It is concluded that the herbicides, paraquat and 2,4-D are hepatotoxic and initiate the process of cell death by decreasing cellular GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of 4-methoxymandelic acid by lignin peroxidase. Mediation by veratryl alcohol

The mechanism of veratryl alcohol-mediated oxidation of 4-methoxymandelic acid by lignin peroxidase was studied by kinetic methods. For monomethoxylated substrates not directly oxidized by lignin peroxidase, veratryl alcohol has been proposed to act as a redox mediator. Our previous study showed that stimulation of anisyl alcohol oxidation by veratryl alcohol was not due to mediation but rather due to the requirement of veratryl alcohol to complete the catalytic cycle. Anisyl alcohol can react with compound I but not with compound II. In contrast, veratryl alcohol readily reduces compound II. We demonstrate in the present report that the oxidation of 4-methoxy mandelic acid is mediated by veratryl alcohol. Increasing veratryl alcohol concentration in the presence of 2 mM 4-methoxymandelic acid resulted in increased oxidation of 4-methoxymandelic acid yielding anisaldehyde. This is in contrast to results obtained with anisyl alcohol where increased concentrations of veratryl alcohol caused a decrease in product formation. ESR spectroscopy demonstrated that 4-methoxymandelic acid caused a decrease in the enzyme-bound veratryl alcohol cation radical signal, which is consistent with its reaction at the active site of the enzyme.     

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.     

Localization of nervonic acid beta-oxidation in human and rodent peroxisomes: impaired oxidation in Zellweger syndrome and X-linked adrenoleukodystrophy

Studies with purified subcellular organelles from rat liver indicate that nervonic acid (C24:1) is beta-oxidized preferentially in peroxisomes. Lack of effect by etomoxir, inhibitor of mitochondrial beta-oxidation, on beta-oxidation of lignoceric acid (C24:0), a peroxisomal function, and that of nervonic acid (24:1) compared to the inhibition of palmitic acid (16:0) oxidation, a mitochondrial function, supports the conclusion that nervonic acid is oxidized in peroxisomes. Moreover, the oxidation of nervonic and lignoceric acids was deficient in fibroblasts from patients with defects in peroxisomal beta-oxidation [Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD)]. Similar to lignoceric acid, the activation and beta-oxidation of nervonic acid was deficient in peroxisomes isolated from X-ALD fibroblasts. Transfection of X-ALD fibroblasts with human cDNA encoding for ALDP (X-ALD gene product) restored the oxidation of both nervonic and lignoceric acids, demonstrating that the same molecular defect may be responsible for the abnormality in the oxidation of nervonic as well as lignoceric acid. Moreover, immunoprecipitation of activities for acyl-CoA ligase for both lignoceric acid and nervonic acid indicate that saturated and monoenoic very long chain (VLC) fatty acids may be activated by the same enzyme. These results clearly demonstrate that similar to saturated VLC fatty acids (e.g., lignoceric acid), VLC monounsaturated fatty acids (e.g., nervonic acid) are oxidized preferentially in peroxisomes and that this activity is impaired in X-ALD. In view of the fact that the oxidation of unsaturated VLC fatty acids is defective in X-ALD patients, the efficacy of dietary monoene therapy, "Lorenzo's oil," in X-ALD needs to be evaluated.