A. Bortolozzi

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.

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.

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.

Morphological study of 5-HT neurons and astroglial cells on brain of adult rats perinatal or chronically exposed to 2,4-dichlorophenoxyacetic acid.

2,4-D is a chlorophenoxyherbicide used worldwide. We have studied the morphological alterations of 5-HT neurons and glial cells in the mesencephalic nuclei of adult rats exposed to 2,4-D both perinatally (during pregnancy and lactation) and chronically (during pregnancy, lactation and after weaning) with quantitative methods. Pregnant rats were daily exposed to 70 mg/kg of 2,4-D from gestation day (GD) 16 to post-natal day (PND) 23 through diet. After weaning, pups were assigned to one of two sub-groups: T1 (fed with untreated diet until PND 90) and T2 (maintained with 2,4-D diet until PND 90). Brain sections were immunocytochemically stained using polyclonal anti-5-HT, anti-GFAP and anti-S-100 protein antibodies as cells markers. 2,4-D exposure during pregnancy and lactancy (T1 group) produced an increase in 5-HT neuronal area and immunoreactivity (IR) in the mesencephalic nuclei studied. However, with the chronical 2,4-D exposure (T2 group) only the 5-HT neuronal area from the dorsal raphe nucleus (DRN) was increased, suggesting an adaptable response of 5-HT neurons in median raphe nucleus (MRN). The presence of reactive astrocytes in mesencephalic nuclei and in hippocampus were also different for the two 2,4-D exposure designs, showing the existence of a correspondence between neuronal changes and astrogliosis. Results support evidences that 2,4-D alters the serotoninergic system and that 5-HT neurons of each mesencephalic nuclei show different responses to the 2,4-D exposure designs which are parallel to astrogliosis.

Increased Sensitivity in Dopamine D2‐like Brain Receptors from 2,4‐Dichlorophenoxyacetic Acid (2,4‐D)‐Exposed and Amphetamine‐Challenged Rats

Abstract: To determine whether the dopamine D2 receptor plays a crucial role in chemically acquired sensitivity to drugs of abuse like amphetamine (AMPH) after an exposure to aryloxoalkanoic compounds, we examined in the present work the impact of AMPH (10 mg/kg, i.p.) on the dopaminergic D2‐like receptors. Rats were exposed to 2,4‐D 70 mg/kg/day 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). After that, an acute challenge with AMPH was administered to each animal. Rats were sacrificed at 0, 5, 24, 72, and 168 h after AMPH, and membranes of striatum (CPu), prefrontal cortex (PfC), hippocampus (H), and cerebellum (Ce) were obtained. Binding studies employing [3H]nemonapride showed that AMPH caused an increase in DA D2‐like receptors of all brain areas between 5 and 24 h after the treatment, with a reduction to the basal levels one week later. The AMPH challenge to (T1 and T2) 2,4‐D‐exposed rats showed an alteration on receptor density depending on brain area and on sex, more than on the 2,4‐D exposure time. This D2‐like receptor density increase could explain the exacerbated behaviors of the 2,4‐D‐exposed and amphetamine‐challenged animals, as previously observed by us. The withdrawal of 2,4‐D did not produce a real reversion to basal levels of D2‐like receptors, indicating that herbicide exposure during the preweanling period caused a sensitization and a stable DA D2‐like receptor increase that was elicited when the system was challenged with this dopaminergic drug.

Intoxication by Non-Protein Nitrogen Compounds In Rat Feed

Last year our white rats (Wistar origin) showed acute behavioral and physiological changes followed by death in 70% of the animals. We detected that the malfunctions could be attributed to the new batch of laboratory rat pellets provided two weeks before. High levels of urea (260 mg/kg) and ammonia (540 mg/kg) were found in the feed while usual values in other similar feed were 48 mg/kg and 82 mg/kg respectively. Suspecting an ammonia intoxication, concentrations of ammonia and urea were determined in blood, brain and liver. Brain neurotransmitters and blood tryptophan and serotonine (5-HT) were also determined. Blood ammonia in rats fed the contaminated feed was about 100% higher than those fed the normal feed while liver and brain ammonia were three and four fold high respectively. Liver and brain urea were four to five fold and about 100% higher in the exposed group than in the group fed the control diet respectively. Blood 5-HT increased 62.33% in females and 99% in males whereas brain 5-HT increased 83.13% in females and 70.47% in males. But, we detected a 59.8% decrease in brain dopamine levels in females and a 38.65% decrease in males. Liver histology showed small droplets of fat stores mainly in centrolobular hepatocyte. No differences in blood or liver cholesterol concentrations were observed whereas liver triacylglycerides were significantly higher in intoxicated females. This study illustrates a problem of food borne intoxication that justifies the need for exhaustive analyses of even not usual compounds in every feed batch; moreover, it is demonstrated that rat behavior appears to be the earliest biomarker of ammonia exposure.