Elsevier

Toxicology

Volume 271, Issues 1–2, 30 April 2010, Pages 13-20
Toxicology

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

https://doi.org/10.1016/j.tox.2010.01.016Get rights and content

Abstract

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 70 mg 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 8 h, returned to their mothers and allowed to suckle for a period of 15 min. 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.

Introduction

A large amount of data indicates that substances naturally present in the environment or of anthropogenic origin (the endocrine disruptors (EDs)) are able to interfere with the functioning of the endocrine system in vertebrates (Crews et al., 2000, Taylor and Harrison, 1999). They may mimic, block, or modulate the synthesis, release, transport, metabolism, binding, or elimination of natural hormones (Gore, 2001). The results of endocrine disruption are often not easily detected. EDs have been shown to disrupt embryonic development, sexual differentiation, reproduction, immune function, behaviour, other responses mediated by hormones (Panzica et al., 2005).

Lactation is the final step of the reproductive cycle. The endocrine control of lactation is a complex physiological process that involves a wide array of hormones, growth factors, neurotransmitters and proteins for the proper regulation of mammogenesis, lactogenesis, galactopoiesis and galactokinesis (Buhimschi, 2004). This provides multiple targets for the action of xenobiotics (EPA/630/R-96/012). Once lactation has been established, it is maintained by the suckling stimulus, that produces the removal of milk from the mammary gland and the secretion of the hormones involved in milk ejection, namely oxytocin, and in milk secretion, mainly prolactin (PRL). Suckling produces a neural signal sent from the nipples to the hypothalamus, where it induces release of oxytocin (OT) and PRL. OT acts by inducing contraction of the myoepithelial cells and voiding of milk, while PRL stimulates milk synthesis (Tucker, 2000). Any modifications produced on these systems could have consequences on the amount and quality of milk produced by the dams. This fact is crucial during lactation because in most mammals, milk is the only source of energy for the pups during this period. Although milk transfer to the litter may not be considered a classical indicator of toxicity, if any environmental contaminant has the capacity of negatively affecting milk transfer to the pups of the mammalian species, man included, that come in contact with it, this certainly can result in detrimental effects on the health and survival of the species. This sole fact justifies the study of the impact of environmental contaminants on lactational performance, independently of whether this effect is due to adverse pharmacological or to toxicological responses.

2,4-Dichlorophenoxyacetic acid is a systemic chlorophenoxy herbicide widely used during the last 60 years to control broadleaf weeds in cereal cropland and on industrial property, lawns, turf, pastures and gardens (Munro et al., 1992). Sales of this herbicide have remained fairly constant in the US and Western Europe over the last decade, but have increased in a number of developing countries, particularly Argentina and Brazil. Exposure to this chemical through agricultural use, food products, or through lawn and garden use has been demonstrated in several studies (Kohli et al., 1974, Taskar et al., 1982, Harris et al., 1992).

Little is known about possible direct effects of 2,4-D during the lactation period, specially on the production and ejection of milk. In previous studies we have demonstrated that 2,4-D is transferred to the neonates through mothers’ milk and causes a dose-dependent decrease on the pup's body weight gain, a reduction on milk content of fatty acids with a chain length of 20 carbons or more and an alteration in the pattern of milk proteins, suggesting that 2,4-D may have some deleterious effect on mammary gland function (Stürtz et al., 2000, Stürtz et al., 2006).

In mammals, PRL release is mainly under inhibitory control exerted by dopamine (DA) originating in the neurons of the arcuate and periventricular nuclei, but a great variety of hypothalamic and non-hypothalamic factors also participate in this regulation. It has been demonstrated that the suckling stimulus results in a decrease of DA released into portal blood (de Greef et al., 1981) that arrives at the anterior pituitary gland (Freeman et al., 2000). In addition, it has also been shown that serotoninergic pathways originating in the raphe nuclei mediate the suckling-induced PRL release (Fessler et al., 1984, Van de Kar and Bethea, 1982, Jahn and Deis, 1994). 5-HT mechanisms of action may involve stimulation of some PRL-releasing factors (Clemens et al., 1978, Garthwaite and Hagen, 1979) or inhibition of the PRL-inhibiting hormone secreted from the hypothalamus (Lynch et al., 1984, Pilotte and Porter, 1981).

On the other hand, nitric oxide (NO) has been shown to play a regulatory role in neuroendocrine function and PRL secretion (Brann et al., 1997). NO inhibits PRL secretion, both in vivo and in vitro (Duvilansky et al., 1995, Lafuente et al., 2004) at anterior pituitary level. It is synthesised from l-arginine by nitric oxide synthase (NOS) (Dawson and Snyder, 1994, Knowles et al., 1989). Within the hypothalamus, NOS activity is found in the paraventricular and supraoptic nuclei as well as in the lamina terminalis (Bhat et al., 1995, Bredt et al., 1991). Moderate activity is found in the medial preoptic nucleus, ventromedial nucleus, suprachiasmatic nucleus, and in median eminence (Bhat et al., 1995).

The goal of the present study was to determine whether 2,4-D alters milk transfer to the litter, and if so, if this alteration could be linked to changes in the serum hormone and brain monoamine levels and/or NOS activities related to these processes.

Section snippets

Animals and exposure to 2,4-D

Nulliparous female rats of Wistar origin, between 90 and 110 days old and weighing approximately 180–210 g were obtained from the animal breeding colony of the Pharmacy and Biochemical Faculty, Rosario, Argentina. Stages of the reproductive cycle were monitored via daily cytological examination of vaginal smears. They were mated individually with fertile males on the night of pro-oestrus. Mating was confirmed by observing sperm in smears on the morning following mating. This day was designated

Clinical examination

Acute or chronic administration of 2,4-D at any dose did not affect adversely the general condition and body weight gain of the dams and any pups died during the test period. The treatments did not affect mean food consumption either. As no significant differences were observed between both control groups of dams, we grouped them together.

Pups body weights

The chronically treated groups (Experiment 1) had a significant reduction (p < 0.001) in body weight, with the greatest effects observed in the 50 and 70 mg

Discussion

The present results clearly show that acute or chronic exposure to increasing doses of 2,4-D produced a significant inhibition in milk transfer to the litter in lactating rats, that may be the main cause of the previously found decreased growth rate of pups born to mothers exposed to this herbicide (Stürtz et al., 2000, Stürtz et al., 2006). Once lactation is established, the rate of milk secretion is adjusted to the demands of the offspring primarily by feedback mechanisms thought to be

Conflict of interest statement

None.

Acknowledgments

This research was supported by Grant to Dra. Ana María Evangelista de Duffard from the OPS/RELAB and the Agencia Nacional de Investigaciones Científicas y Tecnológicas of República Argentina. ROD, RPD, VR and GAJ are career scientists from CONICET.

References (49)

  • G.C. Panzica et al.

    Introduction: neurobiological impact of environmental estrogens

    Brain Res. Bull.

    (2005)
  • N. Stürtz et al.

    Detection of 2,4-dichlorophenoxyacetic acid in rat milk of dams exposed during lactation and milk analysis of their major components

    Food Chem. Toxicol.

    (2006)
  • M.R. Taylor et al.

    Ecological effects of endocrine disruption: current evidence and research priorities

    Chemosphere

    (1999)
  • E. Aguilar et al.

    Interactions between N-methyl-d-aspartate, nitric oxide and serotonin in the control of prolactin secretion in prepubertal male rats

    Eur. J. Endocrinol.

    (1997)
  • Z.B. Andrews et al.

    Dissociation of prolactin secretion from tuberoinfundibular dopamine activity in late pregnant rats

    Endocrinology

    (2001)
  • G.K. Bhat et al.

    Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing hormone neurons and co-localization with N-methyl-d-aspartate receptors

    NeuroEndocrinology

    (1995)
  • D.W. Brann et al.

    Gaseous transmitters and neuroendocrine regulation

    NeuroEndocrinology

    (1997)
  • D.S. Bredt et al.

    Isolation of nitric oxide synthase, a calmodulin-requiring enzyme

    Proc. Natl. Acad. Sci.

    (1990)
  • A.E. Calogero et al.

    Effect of selective serotonin agonists on basal, corticotrophin-releasing hormone- and vasopressin-induced ACTH release in vitro from rat pituitary cells

    J. Endocrinol.

    (1993)
  • A.E. Calogero et al.

    Role for serotonin3 receptors in the control of adrenocorticotropic hormone release from rat pituitary cell cultures

    Eur. J. Endocrinol.

    (1995)
  • J.A. Clemens et al.

    Evidence that serotonin neurons stimulate secretion of prolactin releasing factor

    Life Sci.

    (1978)
  • D. Crews et al.

    Endocrine disruptors: present issues, future directions

    Q. Rev. Biol.

    (2000)
  • W.J. de Greef et al.

    Dopamine levels in hypophysial stalk plasma and prolactin levels in peripheral plasma of the lactating rat: effects of a simulated suckling stimulus

    NeuroEndocrinology

    (1981)
  • T.M. Dawson et al.

    Gases as biological messengers: nitric oxide and carbon monoxide in the brain

    J. Neurosci.

    (1994)
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