Ca2+ homeostasis alterations induced by 2,4-dichlorophenoxyacetic butyl ester and 2,4-dichlorophenoxyacetic acid on avian skeletal muscle.

Fertilized hen eggs were treated externally with 2,4-dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) before the start of the incubation. Actomyosin and sarcoplasmic reticulum adenosine triphosphatase (ATPase) activities from leg and complexus muscles of chicks hatched from treated eggs were measured. No significant variations were detected in the ATPase activities of actomyosin, but the sarcoplasmic reticulum Mg2(+)-activated ATPase and Ca2+, Mg2(+)-activated ATPase were inhibited 50 and 38% respectively. 45Ca2+ uptake into soleus muscle was increased by the 2,4-D b.e. treatment. The compartmental analysis of 45Ca2+ uptake kinetics showed increases in Ca2+ fluxes in sarcolemma and mitochondria and in the mitochondrial calcium pool. Isolated soleus muscles were treated with 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D b.e. [14C]2,4-D reached it highest level in these muscles after 1 hr of treatment. The in vitro treatment with 2,4-D or 2,4-D b.e. increased 45Ca2+ uptake into the muscles. 2,4-D b.e. produced greater alterations than 2,4-D. The compartmental analysis of the 45Ca2+ uptake kinetics also showed increases of the mitochondrial Ca2+ pool and Ca2+ fluxes through sarcolemma and mitochondria. These results led to a hypothesis based on Ca2+ permeability alterations for explaining the myopathic actions of these phenoxyherbicides.

Biochemical alterations in skeletal muscle induced by 2,4-dichlorophenoxyacetic butyl ester during chick embryonic development.

2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) was applied on fertile hen eggs before starting the incubation. Chicks hatched from treated eggs showed motor dysfunctions, postural troubles and edematous muscles. The electromyography revealed muscular weakness, prolonged motor distal latency, and myotonia. The biochemical composition of leg and complexus muscles from 1-day-old chicks was determined. A significant diminution (24%) in the glycogen level of leg muscles was produced by the treatment. There was a small increase (15%) in sarcoplasmic proteins from leg muscles and an increase of a 20 kD protein in the myofibrillar proteins from complexus muscles. Even though total lipid content was not changed, 2,4-D b.e. treatment produced a diminution of sterol esters (20%) and phosphatidylcholine (11%) and an increase of phosphatidylserine (61%), triglycerides (37%) and free fatty acids (FFA) (448%) in leg muscles. Increases of phosphatidylethanolamine (16%), sterols (58%) and FFA (267%) were detected in complexus muscles. A remarkable increase (700-1500%) of unsaturated FFA, e.g. oleic, linoleic and arachidonic acids, was observed. Considering the avian embryo lipid metabolism, it is proposed that FFA and triglycerides were accumulated because they could not be metabolized in the mitochondria. Since FFA are potent cytotoxic compounds, their increase may be a key factor in the 2,4-D b.e. toxic action in muscle and other tissues during embryonic development.

2,4-Dichlorophenoxyacetic acid disrupts the cytoskeleton and disorganizes the Golgi apparatus of cultured neurons.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a potent neurotoxic herbicide widely used in agriculture. The basic mechanisms by which 2,4-D produces cell damage have not yet been determined. In this study we have examined the effects of 2,4-D in primary cultures of cerebellar granule cells in order to obtain insights into the possible mechanisms underlying the toxic effects of this herbicide. The results obtained indicate that a 24-hour exposure to 2,4-D produces a striking and dose-dependent inhibition of neurite extension. This phenomenon is paralleled by a significant reduction in the cellular content of both dynamic and stable microtubules, a disorganization of the Golgi apparatus, and an inhibition in the synthesis of complex gangliosides. Interestingly, 2,4-D inhibits the in vitro polymerization of purified tubulin. Taken together, the present observations raise the possibility that at least one basic mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly. That event may cause a decreased neurite outgrowth response, and could also explain the observed differences in the pattern of ganglioside biosynthesis and/or the disorganization of the Golgi apparatus.

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.

Chick brain hypomyelination produced by 2,4-dichlorophenoxyacetic butyl ester treatment.

Fertilized hens' eggs were externally treated with 2,4-dichlorophenoxyacetic (2,4-D) butyl ester (2.4 mg/egg) before starting incubation. The specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) was diminished in the whole brain of animals born from treated eggs, as compared with controls. Myelin was isolated by ultracentrifugation from the central nervous system. Treatment with 2,4-D butyl ester produced a 65% reduction. There were no statistically significant differences in myelin phospholipids, cerebrosides, sulphatides and gangliosides between control and treated animals. Cholesterol was diminished by 12% in the treated group, while cholesterol esters were not detectable in either myelin fraction. Total myelin proteins were also decreased by 40%, without variations in their pattern. There were changes in the following ratios in the myelin of the treated group: lipid/protein, cholesterol/phospholipids, cholesterol/protein, phospholipids/protein. Collectively these findings indicate that 2,4-D butyl ester produced hypomyelination.

Regionally specific changes in central brain monoamine levels by 2,4-dichlorophenoxyacetic acid in acute treated rats.

As reported in our previous studies, "Serotonin Syndrome", rearing and circling behaviors, were the responses of rats treated with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and challenged with amphetamine. An altered serotonergic and dopaminergic system may be responsible for these behaviors. In this work, endogenous levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) were measured in the areas of the mesocortico-limbico and nigrostriatal systems of rats treated with 50 or 100 mg/kg of 2,4-D, but also challenged with amphetamine (5 or 10 mg/kg), in order to determine regional brain contents of biogenic amines and their metabolites and to find a correlation with the behaviors described. The prefrontal cortex (PFc), striatum, nucleus accumbens (NA), midbrain, ventral tegmental area (VTA), substantia nigra (SN) and cerebellum were assayed. The herbicide 2,4-D increased the 5-HT and DA basal levels and these increments were dependent on sex, brain area and 2,4-D doses. The challenge with 5 or 10 mg/kg amphetamine to 2,4-D (50 or 100 mg/kg) treated rats also resulted in an area-, sex- and dose-dependent alterations of the 2,4-D effect on dopaminergic and serotonergic systems. We are demonstrating a correlation between rat behavior altered by the herbicide's treatment and the contents of 5-HT and DA in brain areas, such as PFc, VTA, SN, striatum, NA, midbrain and cerebellum. In order for 2,4-D to produce the neurobehavioral alterations, not only are high levels of 5-HT and DA necessary, but these neurotransmitters must be released.

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.

2,4-Dichlorophenoxyacetic acid in developing rats alters behaviour, myelination and regions brain gangliosides pattern.

Neonate rats were exposed to 100 mg/kg from the PND 7 to PND 25 or at 70 mg/kg from PND 12 to PND 25. Treated and control pups were subjected to several behavioral tests (righting reflex, negative geotaxy, forelimbs support and open field) during the period of treatment. At PND 25 the regional effects of 2,4-D on gangliosides composition and myelin deposition were determined. The results indicate that the first design of exposure to 2,4-D produces significant diminutions in body and brain weight from PND 21. Furthermore, these pups showed decrease in GM1 level, diminution in myelin deposition and alterations in all behavioral tests. On the other hand, when treatment was not too severe (minor dose and shorter period of treatment), pups showed alterations in forelimb support and in open field tests without body or brain weight modifications. They also presented diminutions in GM1, mayoritary ganglioside of myelin, and in myelin deposition. These results suggest that in this latter 2,4-D exposure design, undernutrition could not be involved.

Behavioral alterations induced in rats by a pre- and postnatal exposure to 2,4-dichlorophenoxyacetic acid.

The purpose of this study was to determine whether the behavioral development pattern was altered by a pre- and postnatal exposure to 2,4-Dichlorophenoxyacetic acid (2,4-D). Pregnant rats were daily orally exposed to 70 mg/kg/day of 2,4-D from gestation day (GD) 16 to postnatal day (PND) 23. After weaning, the pups were assigned to one of the two subgroups: T1 (fed with untreated diet until PND 90) and T2 (maintained with 2,4-D diet until PND 90). Effects on offsprings were evaluated with a neurotoxicological test battery. Neuromotor reflexes, spontaneous motor activity, serotonin syndrome, circling, and catalepsy were analyzed during various postnatal ages. 2,4-D neonatal exposure induced delay of the ontogeny of righting reflex and negative geotaxis accompanied by motor abnormalities, stereotypic behaviors (excessive grooming and vertical head movements), and hyperactivity in the open field. Adult rats of both sexes (T2 group) showed a diminution of ambulation and rearing, while excessive grooming responses were only observed in T2 males. Besides, these animals manifested serotonin syndrome behaviors, catalepsy, and right-turning preference. Some behaviors were reversible, but others were permanent, and some were only expressed after pharmacological challenges.

Dopamine-beta-hydroxylase immunohistochemical study in the locus coeruleus of neonate rats exposed to 2,4-dichlorophenoxyacetic acid through mother's milk.

Dopamine-beta-hydroxylase (DbetaH), the enzyme that synthesizes noradrenaline from dopamine, was studied in the locus coeruleus (LC) of neonate rats exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through lactation for 14 days (from PND 9 to 22). Pups (22 days old) were anesthetized and fixed by transcardiac perfusion. Control and treated serial sections from brain stem--which correspond with the LC according to the Paxinos and Watson atlas--were simultaneously processed by an immunohistochemistry method for the DbetaH detection. Using an image analysis system, the immunostaining optical density (OD) was measured as an estimation of the enzyme content, and an OD significant decrease in the LC of 2,4-D-exposed animals was observed. As tyrosine hydroxylase levels in the LC are regulated by serotonin and in a previous study we demonstrated that this neurotransmitter was increased in 2,4-D-exposed pups, an indirect effect through serotonergic inhibition could be involved in the decreased DbetaH synthesis in the LC of these pups.

Penetration studies and residue determinations of 2,4-dichlorophenoxyacetic acid butyl ester in fertile hen eggs and chicks hatched from treated eggs.

Fertilized hen eggs were topically applied with 2,4-Dichlorophenoxyacetic acid butyl ester (3.1 mg/egg) before starting incubation. During the incubation time 20% of the compound applied on eggs was lost by volatilization. At different incubation times (0, 1, 5, 10 and 15 days) the wash-off technique was applied to fertile hen eggs and residue analyses of 2,4-D ester were determined and quantified by gas chromatography with an electron capture detector. We found that 2,4-D ester began to be detectable at the embryo from the 5th incubation day and the amount of the compound progressively increased during chick embryonic development. Furthermore chicks hatched from treated eggs showed the presence of the compound in all studied tissues and the highest levels were found in organs such as brain and kidney.

Vulnerability of myelin development of the chick to the herbicide 2,4-Dichlorophenoxyacetic butyl ester.

Fertilized hens' eggs were treated externally with 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) immediately before starting incubation, and after different times of incubation (5, 10 and 15 days). Controls were treated externally with ether. Hatchability studies demonstrated that fetotoxic effects of 2,4-D b.e. were similar on the 0, 5 and 10 incubation day, but the 15 Day Group improved the hatching percentage. One day after hatching, chicks were decapitated, and CNS tissue was dissected. Myelin markers, as cerebrosides and CNP, were determined in cerebrum, cerebellum, brain stem and spinal cord of the four groups. They were reduced in cerebrum and brain stem of the 0, 5 and 10 Day Groups, but in the 15 Day Group they were in normal levels. Cerebellum presented normal myelin marker contents in each group studied, while spinal cord only presented decreased marker contents in the 5 Day Group. UDP galactose-ceramide galactosyl transferase (EC 2.4.1.45) activity was reduced in whole brain of chicks born from eggs treated preincubation. The results show the importance of time drug application and suggest that the vulnerable period in CNS development includes proliferation and development of myelin forming cells. Among CNS regions, cerebrum and brain stem seem to be the most vulnerable to the toxic action of 2,4-D b.e. in the chick.

Nucleic acid content and residue determination in tissues of chicks born from 2,4-dichlorophenoxyacetic butyl ester treated eggs.

The effects of 2,4-dichlorophenoxyacetic butyl ester (2,4-D b.e.) on protein and nucleic acid contents of different tissues of chicks born from externally treated hen's eggs were studied. Residues of 2,4-D b.e. were also determined and quantified by gas liquid chromatography (GLC). A diminished DNA content was observed in skeletal muscle and stomach, an increased one was found in cerebrum and cerebellum, whereas no variations were observed in the other analysed tissues. Protein levels were found decreased in kidney, cerebrum and heart. Besides, the GLC determinations revealed the presence of the 2,4-D b.e. in all the studied tissues finding the highest concentrations in kidney. 2,4-D did not appear to be a significant metabolite of 2,4-D b.e. in this experimental system. The dissimilar effects of 2,4-D b.e. on protein and nucleic acid contents of the studied tissues would be related to a different tissue susceptibility to the drug and/or to drug distribution.