Effect of di(2-ethylhexyl) phthalate on the neuroendocrine regulation of reproduction in adult male rats and its relationship to anxiogenic behavior: Participation of GABAergic system.

The endocrine disruptor di-(2-ethylhexyl) phthalate (DEHP) is used in a variety of consumer products made with polyvinyl chloride and also in the manufacture of medical devices. DEHP disrupts reproductive tract development in an antiandrogenic manner and also may induce neurobehavioral changes. The aim of this study was to investigate the effects of chronic postnatal exposure to DEHP (30 mg/kg body weight/day, orally from birth to day 60) on the neuroendocrine regulation of the gonadal axis and its impact on the anxiety-like behavior in adult male rats, as well as the probable participation of the GABAergic system in these effects. DEHP produced a significant increase in plasmatic luteinizing hormone and follicle stimulating hormone, as well as significant testosterone decrease, accompanied with a decrease in hypothalamic gamma-aminobutyric acid (GABA) concentration. On the other hand, DEHP increased the anxiety-like behavior in the elevated plus maze test, evidenced by a significant decrease in the percentages of time spent in the open arms and the frequency in the open arm entries and a significant increase in the percentage of time spent in closed arms. Neuroendocrine and behavioral effects were reversed by GABA agonists, muscimol (2 mg/kg i.p. ) and baclofen (10 mg/kg i.p.). In conclusion, chronic DEHP postnatal exposure induced a disruption in the neuroendocrine regulation of the testicular axis in young adult male rats, and this effect was correlated with an anxiety-like behavior. Since GABA agonists reversed these effects, the results suggest that GABA could participate in the modulation of reproductive and behavioral DEHP effects.

Different effects by sex on hypothalamic-pituitary axis of prepubertal offspring rats produced by in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP).

This study investigated the effect of pre and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in prepubertal male and female rats. DEHP at doses of 3 and 30mg/kgbw/day was administered orally in the drinking water to dam rats since pregnancy onset until the moment of pups sacrifice at 15 days of age. In these animals gonadotropin serum level and the hypothalamic contents of the amino acids aspartate, glutamate and gamma-aminobutyric acid were determined. No changes in gonadotropin levels and amino acid neurotransmitters were detected at the low dose in both sexes. However, DEHP administered at high dose (30mg/kgbw/day) to dams produced a significant decrease in the inhibitory neurotransmitter GABA and an increase in the stimulatory neurotransmitter aspartate in prepubertal male offspring rats. These modifications were accompanied by gonadotropin serum levels increase. On the contrary, in treated female rats this chemical increased both, aspartate and GABA, which exert a characteristic stimulatory action on gonadotropin in 15-day-old normal females. This study provides new data about changes produced by DEHP on the hypothalamic amino acid neurotransmitters involved in the neuroendocrine reproductive regulation, in prepubertal male and female rat offspring from dams exposed during gestational and lactational periods. These alterations induced by DEHP exposure could be related to the gonadotropin modifications also described in this work, and with changes in the production of sexual hormones previously reported by other authors.

Aging-induced changes in 24-h rhythms of mitogenic responses, lymphocyte subset populations and neurotransmitter and amino acid content in rat submaxillary lymph nodes during Freund's adjuvant arthritis.

In young (two months) and aged (18 months) male rats injected s.c. with Freund's adjuvant or adjuvant's vehicle 18 days earlier, 24-h variations in mitogenic responses, lymphocyte subsets and monoamine and amino acid content were examined in submaxillary lymph nodes. Mitogenic responses to concanavalin A (Con A) and lipopolysaccharide (LPS) were higher during the light phase of daily photoperiod. Old rats exhibited a suppressed or impaired mitogenic response to Con A but not to LPS. Acrophases of 24-h rhythm in lymphocyte subset populations in submaxillary lymph nodes were: 18:37-19:44h (B cells), 09:00-10:08h (T and CD4(+) cells) and 12:19-15:58h (CD8(+) cells). Aging augmented B cells and decreased T, CD4(+) and CD8(+) cells. Significant correlations were found between Con A activity and T cells, between lymph node 5HT content and B, T and CD8(+) lymphocytes, and between lymph node 5HT and taurine and GABA content. Aging increased lymph node 5HT content but did not modify NE content. Lymph node concentration of aspartate, glutamate and taurine was higher at night while that of GABA attained peak values at late afternoon. Old rats injected with Freund's adjuvant showed a higher mean value (glutamate) and smaller amplitude (glutamate, taurine) than their respective young controls. The results further document the effects of aging on the chronobiology of the immune system.

Effect of superior cervical ganglionectomy on 24-h variations in hormone secretion from the anterior hypophysis and in hypothalamic monoamine turnover during the preclinical phase of Freund's adjuvant arthritis in rats.

The effect of superior cervical ganglionectomy (SCGx) on 24-h rhythms of circulating adrenocorticotropic hormone (ACTH), growth hormone (GH), prolactin and luteinizing hormone (LH) and of hypothalamic noradrenaline content and dopamine and serotonin turnover, was assessed in rats 3 days after administering Freund's complete adjuvant. In sham-operated rats, Freund's adjuvant injection increased serum ACTH without affecting its diurnal rhythmicity. SCGx, performed 10 days earlier, suppressed 24-h rhythmicity and augmented mean values of circulating ACTH. A depressive effect of immunization on GH release was found in both sham-operated and SCGx rats. GH concentrations did not exhibit diurnal rhythmicity and decreased after immunization. Time-of-day-related changes in serum prolactin were significant for all examined groups, except for SCGx-immunized rats. Freund's adjuvant administration augmented prolactin secretion. Daily changes in serum LH concentration and a decrease after immunization were found in both sham-operated and SCGx rats. SCGx: (i) counteracted inhibition of daily variations of noradrenaline content in medial hypothalamus of Freund's adjuvant-injected rats; (ii) decreased anterior hypothalamic dopamine turnover and augmented it in the medial hypothalamus; (iii) lowered amplitude of serotonin turnover rhythm in medial hypothalamus. The data indicate that several early changes in levels and 24-h rhythms of circulating ACTH and prolactin, and in hypothalamic noradrenaline content and dopamine and serotonin turnover, were modified by a previous SCGx in Freund's adjuvant-injected rats.

Effect of melatonin on bone metabolism in ovariectomized rats.

To assess the effect of pharmacological dose of melatonin on bone metabolism in ovariectomized rats, urinary deoxypyridinoline (a marker of bone resorption) and calcium excretion, circulating levels of calcium, phosphorus and bone alkaline phosphatase activity (a marker of bone formation), and bone mineral density (BMD), mineral content (BMC) and bone area (BA) of total body, were measured in adult rats for up to 60 days after surgery. Rats received melatonin in the drinking water (25 microg/ml water) or drinking water alone. Urinary deoxypyridinoline increased significantly after ovariectomy by 51% (30 days after surgery) and by 47% (60 days after surgery). The increase in urinary deoxypyridinoline found 30 days after ovariectomy was not observed in melatonin-treated rats. Urinary calcium concentration was similar in the 4 experimental groups studied, as was the circulating calcium concentration at every time interval examined. Fifteen days after surgery, a significant increase in serum phosphorus and bone alkaline phosphatase levels occurred in ovariectomized rats receiving melatonin as compared to their controls. Sixty days after surgery BMD, BMC and BA decreased significantly in ovariectomized rats, an effect not modified by melatonin. Serum estradiol decreased significantly by 30 days after ovariectomy to attain values close to the limit of detection of the assay by 60 days after ovariectomy. The results support the conclusion that a pharmacological amount of melatonin modifies bone remodeling after ovariectomy and that the effect may need adequate concentrations of estradiol.

Age-dependent changes in 24-hour rhythms of thymic and circulating growth hormone and adrenocorticotropin in rats injected with Freund's adjuvant.

OBJECTIVE: To analyze the 24-hour changes in thymic and serum concentration of growth hormone (GH) and adrenocorticotropin (ACTH) and their correlation with thymic concentrations of glutamate, aspartate, taurine and GABA in young and old rats during the acute phase of adjuvant's arthritis. METHODS: Young (50-day-old) and old (18-month-old) rats were injected subcutaneously with Freund's adjuvant or its vehicle (paraffin oil containing 15% mannide monooleate). Eighteen days later, they were killed at six different time intervals throughout a 24-hour cycle. Serum and thymic levels of GH and ACTH were measured by radioimmunoassay. Thymic amino acid concentration was measured by HPLC. A quantitative assessment of arthritis was made in an independent group of rats by plethysmography. RESULTS: Old rats injected with Freund's adjuvant exhibited fewer clinical signs of inflammation than young rats. Significant 24-hour changes in thymic and serum GH occurred, except for serum GH in adjuvant's vehicle-treated old rats. Aging augmented thymic GH and decreased serum GH. Immunization with Freund's adjuvant did not modify GH concentration. Thymic and serum concentration of GH correlated negatively. Thymic ACTH varied significantly over 24 h with maxima during the dark phase, except in Freund's adjuvant-treated young rats. Maximal serum ACTH levels occurred in the late afternoon except in Freund's adjuvant-treated old rats which showed maxima at night. Immunization with Freund's adjuvant augmented thymic and circulating concentrations of ACTH. Thymic and serum concentration of ACTH correlated positively. Thymic concentration of glutamate, aspartate and taurine decreased in aged rats and correlated significantly with thymic ACTH. CONCLUSION: The results support the existence of a thymic compartment of GH and ACTH that may be independently regulated.

Auditory deprivation modifies biological rhythms in the golden hamster.

To assess to what extent auditory sensory deprivation affects biological rhythmicity, sleep/wakefulness cycle and 24 h rhythm in locomotor activity were examined in golden hamsters after bilateral cochlear lesion. An increase in total sleep time as well as a decrease in wakefulness (W) were associated to an augmented number of W episodes, as well as of slow wave sleep (SWS) and paradoxical sleep (PS) episodes in deaf hamsters. The number of episodes of the three behavioural states and the percent duration of W and SWS increased significantly during the light phase of daily photoperiod only. Lower amplitudes of locomotor activity rhythm and a different phase angle as far as light off were found in deaf hamsters kept either under light-dark photoperiod or in constant darkness. Period of locomotor activity remained unchanged after cochlear lesions. The results indicate that auditory deprivation disturbs photic synchronization of rhythms with little effect on the clock timing mechanism itself.

Effect of melatonin treatment on 24-h variations in responses to mitogens and lymphocyte subset populations in rat submaxillary lymph nodes.

Wistar male rats were injected s.c. with melatonin (30 microg) or vehicle, 1 h before lights off, for 11 days. Ten days after beginning melatonin treatment, rats received Freund's complete adjuvant or its vehicle s.c., and after 2 days, they were sacrificed at six different time intervals throughout a 24-h cycle. The mitogenic effect of lipopolysaccharide (LPS) and concanavalin A (Con A), the activity of ornithine decarboxylase (ODC) and the relative size of lymphocyte subset populations were measured in submaxillary lymph nodes. In control rats, the mitogenic effects of LPS and Con A and ODC activity peaked during the afternoon. Injection of Freund's adjuvant induced a 10-h shift in the diurnal rhythm of the mitogenic effect of LPS to attain maximal values at night. Melatonin pretreatment blunted the daily variations in the mitogenic activity of Con A or LPS and, when given to Freund's adjuvant-injected rats, augmented mesor and amplitude of diurnal rhythm in ODC activity. Maxima in B cell number occurred at night whereas those of T and B-T cell number occurred during the afternoon. During the early phase of immunization tested, the number of B cells augmented and the amplitude of its diurnal rhythmicity increased both after immunization and following melatonin pretreatment. Maxima of 24-h rhythms in CD4+ and CD4+/CD8+ cell populations occurred during the afternoon while those of CD8+ cells occurred at late night. Melatonin significantly augmented CD4+ cell number and decreased CD8+ cell number; it therefore augmented the CD4+:CD8+ ratio. The results suggest that pretreatment with a pharmacological dose of melatonin exerts immunomodulating effects at an early, preclinical, phase of Freund's adjuvant-induced arthritis in rats.

Psychoimmune neuroendocrine integrative mechanisms revisited.

This review analyzes recent publications on the topic of psycho-immune-neuroendocrine integrative mechanisms. Results on the role of cytokines in cognitive processes and in a major neuroendocrine event, i.e. the release of gonadotropin-releasing hormone, are discussed, as are the effects of cytokines on central neurotransmission. The control of immune responses by local sympathetic nerves, a major pathway in neuroimmune communication, is discussed. This review also updates information indicating that melatonin is a circulating signal affecting the periodic organization of the immune response.

Effect of pinealectomy, superior cervical ganglionectomy, or melatonin treatment on 24-hour rhythms in ornithine decarboxylase and tyrosine hydroxylase activities of rat spleen.

Diurnal variations in splenic ornithine decarboxylase and tyrosine hydroxylase activities were examined in rats subjected to pinealectomy, bilateral superior cervical ganglionectomy, or their respective sham operations. Rats were treated with Freund's complete adjuvant or its vehicle 2 days before sacrifice. After immunization, splenic ornithine decarboxylase activity was augmented 5-6-fold. In both immunized and nonimmunized sham-operated rats, significant diurnal variations in ornithine decarboxylase activity were detectable, with a maximum at early morning, acrophases after Cosinor analysis varying from 0845 to 1048h. In pinealectomized or superior cervical ganglionectomized, immunized rats, ornithine decarboxylase activity attained values 22-27% lower than those of immunized sham-operated controls, while amplitude decreased significantly by 27-30%. Administration of melatonin (30 microg/animal s.c. at late evening for 11 days in immunized rats) significantly augmented mesor levels of splenic ornithine decarboxylase activity and increased the amplitude of the diurnal rhythm both in pinealectomized and in superior cervical ganglionectomized rats. Melatonin treatment also augmented rhythm mesor in immunized, sham-ganglionectomized rats, as well as rhythm amplitude in immunized and nonimmunized, sham-ganglionectomized rats. Splenic tyrosine hydroxylase activity attained its maximum at late afternoon and early night, with acrophases varying from 1800 to 2023h. Immunization significantly increased mesor values of splenic tyrosine hydroxylase activity, whereas neither pinealectomy nor superior cervical ganglionectomy affected circadian rhythm parameters. Melatonin treatment augmented mesor values of tyrosine hydroxylase rhythm and increased its amplitude in pinealectomized, ganglionectomized, or sham-operated rats. The results are compatible with the view that the pineal gland plays a role in circadian changes of immune responsiveness in rat spleen via an immunopotentiating effect of melatonin on splenic cell proliferation.

Diurnal rhythms in ornithine decarboxylase activity and norepinephrine and acetylcholine synthesis and acetylcholine synthesis of rat submaxillary lymph nodes: effect of pinealectomy, superior cervical ganglionectomy and melatonin replacement.

The effect of pinealectomy, superior cervical ganglionectomy and melatonin replacement on diurnal variations in submaxillary lymph node ornithine decarboxylase activity, tyrosine hydroxylase activity and [3H]choline conversion to [3H]acetylcholine were examined in rats subjected to pinealectomy, bilateral superior cervical ganglionectomy or their respective sham-operations, and treated with Freund's complete adjuvant or its vehicle. In both immunized and nonimmunized sham-operated rats, significant diurnal variations in ornithine decarboxylase activity were detectable, with a maximum at 13.00 h (vehicle) or at 17.00 h (Freund's adjuvant). In rats subjected to pinealectomy, ornithine decarboxylase activity decreased by about half, still exhibiting significant diurnal variations with a maximum at 13.00 h. Abolition of circadian rhythmicity and depression of ornithine decarboxylase activity to about one third of controls were found in submaxillary lymph nodes of bilaterally superior cervical ganglionectomized rats. Administration of melatonin (30 micrograms/animal) in the late evening during 11 days counteracted the depressed levels and suppressed the amplitude of diurnal rhythmicity of ornithine decarboxylase in pinealectomized or bilaterally superior cervical ganglionectomized rats, as well as augmented enzyme activity in sham-operated controls. The amplitude and mean levels of 24-hour rhythms in submaxillary lymph node tyrosine hydroxylase activity and [3H]choline conversion to acetylcholine (that attained their maxima at 21.00-1.00 and 13.00-17.00 h, respectively) decreased significantly after pinealectomy, these effects being significantly counteracted by melatonin injection. Melatonin augmented tyrosine hydroxylase activity and acetylcholine synthesis in sham-pinealectomized rats. The results are compatible with the view that the pineal gland plays a role in circadian changes of immune responsiveness in lymphoid tissue via an immunopotentiating effect of melatonin on lymph node cell proliferation.