Oxidative stress in ventral prostate, ovary, and breast by 2,4-dichlorophenoxyacetic acid in pre- and postnatal exposed rats.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely used in agriculture and forestry since the 1940s. 2,4-D has been shown to produce a wide range of adverse effects-from embryotoxicity and teratogenicity to neurotoxicity-on animal and human health. The purpose of this study was to determine the possible effects of pre- and postnatal exposure to 2,4-D on oxidative stress in ventral prostate, ovary and breast. Pregnant rats were daily exposed to oral doses of 70 mg/kg/day of 2,4-D from 16 days of gestation up to 23 days after delivery. Then, the pups were sacrificed by decapitation at postnatal day (PND) 45, 60, or 90. Antioxidant enzyme activities and some parameters of the oxidative stress were assessed in ventral prostate, breast, and ovary. Results show that 2,4-D produced three different effects. First, it increased the concentration of some radical oxygen species and the rates of lipid peroxidation and protein oxidation in ventral prostate, thereby causing oxidative stress at all ages studied. Although an increase in the activity of some antioxidant enzymes was detected, this seemed to have been not enough to counteract the oxidative stress. Second, 2,4-D promoted the oxidative stress in the breasts, mainly during puberty and adulthood, probably because the developing gland is more sensitive to xenobiotics than the adult organ. Third, 2,4-D altered the activity of some antioxidant enzymes and increased lipid peroxide concentration in the ovary. This effect could reflect the variety of ovarian cell types and their different responses to endocrine changes during development.

Effect of 2,4-dichlorophenoxyacetic acid on milk transfer to the litter and prolactin release in lactating rats.

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on brain monoamines and the serum level of hormones involved in milk synthesis and on the milk ejection reflex in rats were evaluated. Dams were treated with 2.5, 5, 15, 25, 50 or 70mg 2,4-D/kg bw according to two experimental designs: (a) through food from post partum day 1 (PPD 1) to PPD 16 and the respective control groups or (b) an unique i.p. injection on PPD 11. To measure milk ejection, the litter was separated from the mother at the 11th day of lactation during 8h, returned to their mothers and allowed to suckle for a period of 15min. The procedure was repeated on 3 consecutive days until the end of treatment. The change in litter weight during the suckling period was taken as a measure of the amount of milk ejected during this period. The dams' serum prolactin (PRL), oxytocin (OT) and growth hormone levels were determined by radioimmunoassay. Both treatment regimens produced a dose-dependent decrease in the amount of milk ejected and circulating PRL and OT secreted in response to the suckling stimulus. Administration of OT before returning the pups restored the milk ejection, indicating no impairment in the capacity of the mammary gland to produce and secrete milk. In addition, dopamine levels were increased by the 2,4-D treatments in arcuate nucleus (ArN) and anterior lobe of pituitary gland (AL), while serotonin level was drastically decreased in ArN. 2,4-D treatment increased both calcium-dependent and calcium-independent nitric oxide synthase (NOS) activities in ArN. These results suggest that 2,4-D inhibits the suckling-induced hormone release, milk transfer to the litter at the central level, through a stimulation of hypothalamic NOS and dopamine and by an inhibition of hypothalamic serotonin transmission.

Neonatal hypomyelination by the herbicide 2,4-dichlorophenoxyacetic acid. Chemical and ultrastructural studies in rats.

The purpose of this study was to determine whether 2,4-dichlorophenoxyacetic acid (2,4-D), which is an herbicide used to control the growth of broadleaf weeds, had a direct or an indirect (mediated by undernutrition) hypomyelinating effect. We also proposed to analyze the effect of 2,4-D on undernourished (UN) pups. Four experimental rat groups were used: well-nourished (WN) pups, litters with eight offsprings; UN pups, litters with fourteen offsprings; WN pups whose mother received 70 mg/kg/day of 2,4-D from postnatal day (PND) 9 to 21 (WN70 pups); and UN pups whose mother received 70 mg/kg/day of 2,4-D from PND 9 to 21 (UN70 pups). In this work, we demonstrated that (1) myelin proteins (analyzed by Western blot and/or immunohistochemical study) showed a significant decrease in WN70, UN, and UN70 with respect to control group; (2) there is a good correlation between these myelin-specific protein expression with the degree of myelin compaction detected by electron microscopy in groups exposed to 2,4-D; (3) a decreased and normal number of myelin sheets were detected in UN and 2,4-D exposed pups, respectively; and (4) undernourishment sensitized pups to the hypomyelinating effect of 2,4-D. According to this and besides the fact that WN70 group have no body weight changes, these results are indicating that 2,4-D and undernourishment are two independent hypomyelinating factors.

Effect of 2,4-dichlorophenoxyacetic acid on rat maternal behavior.

Exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) has several deleterious effects on the nervous system such as alterations in the concentrations of neurotransmitters in the brain and/or behavioral changes, myelination rate, ganglioside pattern [Bortolozzi, A., Duffard, R., Antonelli, M., Evangelista de Duffard, A.M., 2002. Increased sensitivity in dopamine D(2)-like brain receptors from 2,4-dichlorophenoxyacetic acid (2,4-D)-exposed and amphetamine-challenged rats. Ann. N.Y. Acad. Sci. 965, 314-323; Duffard, R., García, G., Rosso, S., Bortolozzi, A., Madariaga, M., DiPaolo, O., Evangelista de Duffard, A.M., 1996. Central nervous system myelin deficit in rats exposed to 2,4-dichlorophenoxyacetic acid throughout lactation. Neurotoxicol. Teratol. 18, 691-696; Evangelista de Duffard, A.M., Orta, C., Duffard, R., 1990. Behavioral changes in rats fed a diet containing 2,4-dichlorophenoxyacetic butyl ester. Neurotoxicology 11, 563-572; Evangelista de Duffard, A.M., Bortolozzi, A., Duffard, R.O., 1995. Altered behavioral responses in 2,4-dichlorophenoxyacetic acid treated and amphetamine challenged rats. Neurotoxicology 16, 479-488; Munro, I.C., Carlo, G.L., Orr, J.C., Sund, K., Wilson, R.M. Kennepohl, E. Lynch, B., Jablinske, M., Lee, N., 1992. A comprehensive, integrated review and evaluation of the scientific evidence relating to the safety of the herbicide 2,4-D. J. Am. Coll. Toxicol. 11, 559-664; Rosso et al., 2000], and its administration to pregnant and lactating rats adversely affects litter growth and milk quality. Since normal growth of the offspring depends on adequate maternal nursing and care, we evaluated the effect of 2,4-D on rat maternal behavior as well as the dam's monoamine levels in arcuate nucleus (AcN) and serum prolactin (PRL) levels. Wistar dams were exposed to the herbicide through the food from post partum day (PPD) 1 to PPD 7. Dams were fed either with a 2,4-D treated diet (15, 25 or 50mg 2,4-D/kg/daybw) or with a control diet. We observed that maternal nesting behavior was not modified by 2,4-D treatment. However, mother-pup interactions, specially the nursing behavior, were altered. Retrieval, crouching and licking of pups were reduced or suspended after 2,4-D treatment. We also observed an increase in the latency of retrieval and crouching in the dams treated with the herbicide. Dams showed movement along cage peripheries, food consumption during the light phase and high self-grooming. In addition of the deficits observed in maternal behavior parameters, increased catecholamine levels and a drastic decrease in indolamine levels in the AcN of treated dams were determined. Serum PRL levels were also diminished by 62%, 68% and 70% with respect to control dams in the 15, 25 and 50mg 2,4-D/kgbw treated dams, respectively. In conclusion, exposure to 2,4-D during the first post partum days produced changes in maternal behavior, serum prolactin and monoamine levels in the AcN of treated dams.

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.

Selective oxidative stress in brain areas of neonate rats exposed to 2,4-Dichlorophenoxyacetic acid through mother's milk.

2,4-Dichlorophenoxyacetic acid (2,4-D) induced disparate alterations on enzymatic activities of the defensive mechanism and/or modifications of the reactive oxygen species levels in specific neonate rat brain regions. The midbrain, striatum, and prefrontal cortex were the areas where the alterations were more remarkable and with similar tendency. The hippocampus did not suffer many alterations, and the hypothalamus was the area where no changes were observed. The current results suggest that the developing brain areas have different susceptibilities to the adverse effect of the herbicide, especially those areas related to the dopaminergic system, and that oxidative stress is one 2,4-D mechanism of toxicity.

Aryloxoalcanoic compounds induce resistance to antibiotic therapy in urinary tract infection caused by Escherichia coli.

Clofibric acid (CL) is a compound used to control hypertriglyceridemia, and ethacrynic acid (ET) is administered to enhance diuresis. These compounds are structurally analogous to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as they have a chlorinated phenoxy moiety. As these agents are mainly excreted by the renal route, they could potentially coexist with Escherichia coli in the urinary tract of infected patients. Induction of the in vitro resistance of E. coli to hydrophilic antibiotics was determined by increasing the values of the minimum inhibitory concentration (2-40-fold). These results correlated with drastically inhibited expression of the hydrophilic bacterial channel OmpF. In vivo assays were performed in ascending urinary tract infection in female BALB/c mice. Treatment with the hydrophilic antibiotic cephalexin 25 mg kg(-1) day(-1) by the oral route diminished renal infection. The CFU mean values in the kidneys were between 75% and 89% lower than those in animals without treatment. Simultaneous exposure to CL (at a therapeutic dose, 28.6 mg kg(-1) day(-1)) did not change the effect of the treatment. In contrast, ET at 2.9 mg kg(-1) day(-1) or 2,4-D at 70 mg kg(-1) day(-1) inhibited the antibiotic therapeutic effect. Moreover, 2,4-D dramatically increased bacterial infection after 9 days of exposure.

Effects of 2,4-dichlorophenoxyacetic acid exposure on dopamine D2-like receptors in rat brain.

2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been associated with a range of adverse health effects on humans and different animal species. Although the mechanism of 2,4-D neurotoxicity remains unknown, we had previously reported changes in various neurotransmitter systems, such as serotonin (5-HT) and dopamine (DA), which were proposed to mediate some of the behavioral effects in rats. In the present work, we examined the impact of 2,4-D exposure on the ontogeny of dopaminergic D2-type receptors in prefrontal cortex (PFc), striatum (CPu), hippocampus (H) and cerebellum (Cer). Pregnant rats were orally exposed to 70 mg/kg/day of 2,4-D from gestation day (GD) 16 to postpartum day 23. After weaning, the pups were assigned to one of the two subgroups: T1 [fed with untreated diet until postnatal day, (PD) 90] and T2 [maintained with 2,4-D diet until PD 90]. Five to eight pups per age and sex were sacrificed at 6, 15, 30, 45 or 90 days of age for membrane receptor binding assays employing [3H]nemonapride. Subchronic 2,4-D exposure (T2 group) increased DA D2-type receptor around 40% in CPu. In addition, DA D2-type receptor levels also increased in PFc (15 and 30 days) and Cer (30 and 90 days). Sex-dependent differences in D2 receptors were observed with T2 female rats being more affected than T2 male rats. When the herbicide treatment was interrupted after weaning (T1 group), DA D2-type receptor density was apparently recovered and stabilized to control level. These findings suggest a reversible vulnerability of D2-type receptors to 2,4-D exposure. Regional increases of D2-type receptor density may explain certain behaviors reported early by us, such as catalepsy and right-turning preference in rats exposed to 2,4-D.

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 dam's body weight) on iron (Fe), zinc (Zn) and copper (Cu) in brain, liver and serum of well-nourished and undernourished pups exposed through dam's 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 dam's 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.

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.

Environmental chemical compounds could induce sensitization to drugs of abuse.

Chemical environment should be considered as an additional factor that influences drugs of abuse. Besides, maternal exposure to environmental chemicals has increased, and fetuses as well as neonates may be at greater risk than adults. Studies from our laboratory have described a permanent effect of the worldwide use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on serotonin and dopamine content in total brain and specific brain areas of adult rats born of mothers treated during lactation and fed with 2,4-D-treated diet after weaning. These animals show a modified neurotransmitter-related behavioral pattern in their developmental young and in adult age. Drugs that could be used to challenge the dopaminergic or serotoninergic systems include amphetamine or haloperidol, a postsynaptic dopamine receptor blocker. 2,4-D-exposed animals showed exacerbated response to challenges. Postnatal alterations in central dopaminergic and serotoninergic systems due to environmental chemical exposure may contribute to the enhanced/reduced behavioral sensitization to drugs of abuse.

Increased sensitivity in dopamine D(2)-like brain receptors from 2,4-dichlorophenoxyacetic acid (2,4-D)-exposed and amphetamine-challenged rats.

To determine whether the dopamine D(2) 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 D(2)-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 [(3)H]nemonapride showed that AMPH caused an increase in DA D(2)-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 D(2)-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 D(2)-like receptors, indicating that herbicide exposure during the preweanling period caused a sensitization and a stable DA D(2)-like receptor increase that was elicited when the system was challenged with this dopaminergic drug.