Ricardo Duffard

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.

Central nervous system myelin deficit in rats exposed to 2,4-dichlorophenoxyacetic acid throughout lactation

Our results show that 2,4-dichlorophenoxyacetic acid (2,4-D) exposure through mothers milk during the period of rapid myelination (from the 15th to the 25th postnatal days) results in a myelin deficit in the pups brain and demonstrates the vulnerability of the developing central nervous system (CNS) to 2,4-D. After 100 mg/kg 2,4-D administration to dams, brains of male and female rats show a significant diminution of myelin markers such as monohexosylceramide as well as phospholipids and free fatty acids (FFA) and an increase of cholesteryl esters. Histological studies revealed myelin deficit in some brain regions after 2,4-D treatment. These data indicate that 2,4-D, through the mothers milk, alters the myelination process during a specific postnatal period.

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 through lactation induces astrogliosis in rat brain

Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-d-old rat pups exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through the mothers milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocampus and cerebellum. A quantitative analysis of reactive astrocytes was performed by using GFAP and S-100 protein as specific markers. The study showed a significant increase in their number, size, number of processes, and density of immunostaining in 2,4-D-exposed animals. Exposure to 2,4-dichlorophenoxyacetic acid on the first days of life modifies the astroglial cytoarchitecture in parallel to previously described neuronal changes.

Effect of 2,4-dichlorophenoxyacetic acid herbicide on Escherichia coli growth, chemical composition, and cellular envelope

2,4‐Dichlorophenoxyacetic acid (2,4‐D) is a herbicide widely used in the world and mainly excreted by the renal route in exposed humans and animals. Herbicides can affect other nontarget organisms, such as Escherichia coli. We observed that a single exposure to 1 mM 2,4‐D diminished growth and total protein content in all E. coli strains tested in vitro. In addition, successive exposures to 0.01 mM 2,4‐D had a toxic effect decreasing growth up to early stationary phase. Uropathogenic E. coli adhere to epithelial cells mediated by fimbriae, adhesins, and hydrophobic properties. 2,4‐D exposure of uropathogenic E. coli demonstrated altered hydrophobicity and fimbriation. Hydrophobicity index values obtained by partition in p‐xylene/water were 300–420% higher in exposed cells than in control ones. Furthermore, values of hemagglutination titer, protein contents in fimbrial crude extract, and electron microscopy demonstrated a significant diminution of fimbriation in treated cells. Other envelope alterations could be detected, such as lipoperoxidation, evidenced by decreased polyunsaturated fatty acids and increased lipid degradation products (malonaldehyde), and motility diminution. These alterations decreased cell adherence to erythrocytes, indicating a diminished pathogenic capacity of the 2,4‐D‐exposed E. coli. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 43–53, 2001

Melatonin Decreases The Oxidative Stress Produced by 2,4-Dichlorophenoxyacetic Acid in Rat Cerebellar Granule Cells

Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most widely used herbicides due to its relatively moderate toxicity and to its biodegrad-ability in the soil. In toxic concentrations, 2,4- D displays strong neurotoxicity, partly due to generation of free radicals. Since melatonin has remarkable antioxidant properties, the objective of this study was to assess to what extent it was effective in preventing the 2,4-D effect on redox balance of rat cerebellar granule cells (CGC) in vitro. Cellular viability, generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), reduced glutathione (GSH) levels, and the activities of the antioxidant enzymes Cu/ Zn-superoxide dismutase (Cu/Zn-SOD), Mn-SOD, selenium-glutathione peroxidase (Se-GPx) and catalase (CAT) were measured in CGC exposed to 2,4-D and/or melatonin for 48 h. In CGC cultures exposed to 2,4-D, cell viability, GSH levels and CAT activity decreased significantly whereas ROS generation and Se-GPx activities were augmented. Except for Se-GPx activity, all these changes were counteracted by the concomitant addition of 0.1 or 0.5 mM melatonin. In addition, incubation of CGC with melatonin alone resulted in augmentation of cell viability, GSH levels and Se-GPx activity. RNS generation and SOD activity remained unaffected by either treatment. Since melatonin was able to counteract most of redox changes produced by 2,4-D in CGC in culture, the experimental evidence reported further support the efficacy of melatonin to act as a neuroprotector.

Induction of Apoptosis in Cerebellar Granule Cells by 2,4-Dichlorophenoxyacetic Acid

Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) and derivatives are herbicides widely used in Argentina and other parts of the world. Exposure to 2,4-D, its ester and salt formulations, have been associated with a range of adverse health effects in humans and different animal species, from embryotoxicity and teratogenicity to neurotoxicity. In this work, we demonstrate that after 24 hs of treatment with 1 and 2 mM 2,4-D there is an induction of apoptosis in cerebellar granule cells (CGC) in culture. However, with 2 mM 2,4-D one population of CGC developed features of apoptosis while another appeared to die by necrosis. This process is associated with an increase in caspase-3 activity after 12 hs of treatment with the herbicide, which is preceded by cytochrome c release from the mitochondria. Treatment of CGC with 2,4-D appears to induce apoptosis by a direct effect on mitochondria producing cytochrome c release and consequently activation of caspase-3, being mitochondrial damage sufficient for triggering the events that may cause apoptosis.

Intracerebral administration of 2,4-diclorophenoxyacetic acid induces behavioral and neurochemical alterations in the rat brain.

Although, the mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) neurotoxicity remains unknown, the monoaminergic system appears to mediate some of its effects in rats as we previously reported. In this study; we examined the 2,4-D effects on locomotor activity, circling behavior and monoamine levels after the injection into the basal ganglia of male adult rats. These effects were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). 2,4-D-injected into one striatum (100 microg/rat) produced a marked depression in locomotor activity and elicited a moderate circling towards the ipsilateral side at 6 and 24 h postinjection. These behavioral changes were accompanied by a decrease and an increase of serotonin (5-HT) and homovanillic acid (HVA) levels, respectively. 2,4-D administration (100 microg/rat) into the nucleus accumbens, induced similar behavioral and neurochemical patterns to the intrastriatal 2,4-D injection, although rats did not present notorious turning. When 2,4-D was injected into one medial forebrain bundle (MFB, 50 microg/rat), animals presented ipsilateral circling, while locomotor activity was unchanged at 3 and 7 days post-injection. These last rats also exhibited diminished levels of striatal 5-HT, dopamine (DA) and their metabolites without changes in the substantia nigra (SN). Animals sacrificed 3 and 7 days after a 6-OHDA injection into one of the MFB, presented progressive depletion of dopamine in striatum and SN. 2,4-D as well as 6-OHDA-treated rats into one of the MFB were challenged with low dose (0.05 mg/kg s.c.) of apomorphine (only at 7 days post-injection) to evaluate a possible DA-receptor supersensitivity. Only 6-OHDA treated rats showing a vigorous contralateral rotation activity. These results indicate that 2,4-D induced a regionally-specific neurotoxicity in the basal ganglia of rats. The neurotoxic effects of 2,4-D on basal ganglia by interacting with the monoaminergic system depended not only on the exact location of the 2,4-D injection, but also on the dose and time period of post-injection. Toxicity produced by 2,4-D appears to be different in monoaminergic terminals, axonal fibers, and cell bodies.

Iron, zinc and copper levels in brain, serum and liver of neonates exposed to 2,4-dichlorophenoxyacetic acid

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D, 70 or 100 mg/kg dams body weight) on iron (Fe), zinc (Zn) and copper (Cu) in brain, liver and serum of well-nourished and undernourished pups exposed through dams milk were determined. Undernourishment produced a high Fe decrease (serum and brain) and a delay in weight gain similar to that produced by the highest dose of 2,4-D on well-fed pups. In the latter animals, copper was found to be the most altered ion, increasing its level in serum, liver and some brain areas and decreasing in whole brain. Zinc was the most affected ion in brain areas. Well-nourished pups lactationally exposed to 70 mg 2,4-D/kg dams body weight altered neither their metal levels nor their body weight in any of the tissues studied. Undernourished pups were more vulnerable to the 2,4-D effect than well-nourished pups. Undernourished pups exposed to a lower 2,4-D dose showed a decrease in their body, brain and liver weight similar to well-fed animals exposed to 100 mg 2,4-D/kg. A noticeable decrease in liver L-tryptophan peroxidase activity by 2,4-D was also registered. This effect was higher in undernourished and 2,4-D-exposed pups. These results suggest that brain areas have a different susceptibility to the herbicide and that undernourishment produces a higher vulnerability to the herbicide and exacerbates the 2,4-D effect.

Changes in brain serotonin and 5-hydroxyindolacetic acid levels induced by 2, 4-dichlorophenoxyacetic butyl ester☆

Brain concentrations of Serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) were determined in male, mother and virgin female adult rats after exposure to 69 mg/kg body weight/day of 2,4-dichlorophenoxyacetic butyl ester (2,4-Dbe) during 15 or 45 consecutive days. Both 5-HT and 5-HIAA concentrations were increased in the brain. These effects reverted to levels even lower than controls, when the animals were fed an untreated diet after the 2,4-Dbe treatment. High 5-HT and 5-HIAA brain concentrations were also observed in adult rats born from treated mothers (during pregnancy and lactancy) and fed with or without treated diet after weaning. Two different effects on serotoninergic system were detected: a transient effect if 2,4-Dbe was given to adult rats in a short period of time and a permanent effect if the herbicide was supplied during pre- and post birth period (rat brain development). However, in utero exposed but lactationally cross-fostered rat pups were not affected, suggesting that prenatal exposure did not have any influence on the postnatal status of the neurotransmitter(s).