Ana María Evangelista de 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.

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

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.

Effect of the herbicide 2,4-dichlorophenoxyacetic acid on uropathogenic Escherichia coli virulence factors.

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-widely used in the world and mainly excreted by the renal route in exposed humans-were studied on the virulence and surface characteristics of an uropathogenic Escherichia coli strain. When the urine was supplemented with 2,4-D in vitro, the compound significantly reduced the bacterial fimbriation assayed by hemagglutination and surface protein quantification. Protein values decreased from 0.24 mg/g dw to 0.05 or 0.12 mg/g dw by 1 or 0.1 mM 2,4-D treatment, respectively. The effects in vivo were studied in groups of mice challenged intra-urethra with E. coli and exposed by the oral route with three different 2,4-D doses (2.6, 25 or 70 mg/kg bw) during 22 days. Depending on the dose used, the herbicide significantly decreased or removed bacterial cells in mice bladder and kidneys; except in the group treated with the highest dose from the 9th day of treatment. The histological studies showed mononuclear cell infiltration at low doses, and toxic damage in the renal parenchyma at prolonged exposure with higher doses, up to tisular necrosis in the 70 mg/kg bw group after 9 days of treatment. Our investigations performed in an experimental model suggest that short time 2,4-D exposure at low doses could act in prevention of UTI stimulating leukocytic migration and decreasing bacterial fimbriation. On the contrary, high doses and long-term exposure enhanced renal damage resulting in infection recurrence.

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).

Asymmetrical development of the monoamine systems in 2,4-dichlorophenoxyacetic acid treated rats.

The purpose of this study was to determine whether the regional brain biogenic amine levels in adult rats were altered by pre- and post-natal exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). Pregnant rats were daily orally exposed to 70 mg/kg per day of 2,4-D from gestation day (GD) 16 to post-partum day (PPD) 23. After weaning, the pups were assigned to one of two subgroups: T1 fed with untreated diet up to post-natal day (PND) 90 and T2 (maintained with 2,4-D diet up to PND 90). In addition, we wanted to know the effect of 2,4-D on lateralization in the monoamine systems of the basal ganglia of these adult rats and whether there was any correlation with the behavioral developmental pattern previously reported by us. In this study the content of noradrenaline (NA) was significantly increased in substantia nigra (SN) while it decreased in cerebellum in male and female rats of T2 group. The decreased dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) contents in cerebellum, midbrain, ventral tegmental area (VTA) and prefrontal cortex (PFc) showed an alteration in the mesocorticolimbic system. However, an increase of DA in SN and of DOPAC and HVA in nucleus accumbens (NAc) in both sexes and of DA and DOPAC (only in females) in striatum was detected. The contents of serotonin (5-hydroxytryptamine, 5-HT) were significantly increased in both sexes in PFc, striatum (St), midbrain, SN and cerebellum. Variations of any monoamine levels in NAc and VTA were determined. T1 rats were irreversibly altered: a diminution in DA and/or DOPAC levels in PFc, midbrain, VTA and cerebellum was determined. Indolamines of these rats were increased in both sexes in PFc and St. There was also a large increase in 5-HT levels in midbrain of male rats. Although no changes in the dopaminergic system with respect to their control values in any side of these brain structures were observed, DA and DOPAC levels were found to be decreased in the right side with respect to the left side in striata and accumbens nuclei in T2 female rats supporting the behavioral rotation previously registered by us in these rats. In addition, the increased 5-HT content detected in both the right and left striata observed in this study could be the answer to the behaviors observed and to the early alterations in dopamine in basal ganglia by 2,4-D in neonatal exposed rats, mediated by a serotonergic modulation on the dopaminergic system.

Effects of 2,4-dichlorophenoxyacetic acid on central nervous system of developmental rats. Associated changes in ganglioside pattern

Neonate rats were treated with 2,4-dichlorophenoxyacetic acid (2,4-D) from the 7th or 12th until the 17th or 25th postnatal day. Two drug dosages were used: 70 and 100 mg/kg body weight of 2,4-D. At the 17th day of age, no changes were observed in body weight, protein and DNA content. However, 25-day-old treated pups showed diminutions in body and brain weight, protein and DNA levels, depending on doses and period of treatment. With respect to ganglioside levels, few changes were observed in treated animals until the 17th day of age. However, at the 25th day, with higher dose and longer treatment a diminution in all parameters analyzed was observed. These results suggest a delay in CNS development when pups were exposed to a very severe chemical injury with 2,4-D. On the other hand, when the chemical injury was not too severe, the brain would be capable to trigger biochemical mechanisms producing a plasticity response which is expressed as changes in ganglioside content and composition.

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein.

2,4-dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.