Further analysis of behavioral and endocrine consequences of chronic exposure of male Wistar rats to subtoxic doses of endocrine disruptor chlorobenzenes

Physiology & Behavior 103 (2011) 421–430

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Further analysis of behavioral and endocrine consequences of chronic exposure of male Wistar rats to subtoxic doses of endocrine disruptor chlorobenzenes Z. Valkusz a, G. Nagyéri a,⁎, M. Radács b, T. Ocskó b, P. Hausinger c, M. László b, F.A. László c, A. Juhász d, J. Julesz a, R. Pálföldi e, M. Gálfi b a

Endocrine Unit of First Department of Internal Medicine, Faculty of Medicine, University of Szeged, Korányi fasor 8–10, H-6720, Szeged, Hungary Institute of Applied Natural Science, Faculty of Education, University of Szeged, Boldogasszony sugárút 6, H-6725, Szeged, Hungary Department of Physiology, Anatomy and Neuroscience, Faculty of Sciences and Informatics, University of Szeged, Közép fasor 52, H-6726, Szeged, Hungary d Department of Psychiatry, Faculty of Medicine, University of Szeged, Semmelweis utca 6, H-6725, Szeged, Hungary e Department of Pulmonology, Faculty of Medicine, University of Szeged, Alkotmány utca 36, H-6772, Deszk, Hungary b c

a r t i c l e

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Article history: Received 30 November 2010 Received in revised form 1 March 2011 Accepted 10 March 2011 Keywords: Chlorobenzenes Anxiety Aggression Vasopressin Oxytocin Monoamines

a b s t r a c t Many chemicals utilized by humans are present as environmental pollutants and may influence homeostasis from neurological, immunological, endocrinological and/or behavioral aspects. Such agents, acting alone or in ambient mixtures, may be biologically active even at extremely low doses, and it may be postulated that stable, bioaccumulative, reactive endorine disruptors may affect central and/or peripheral secretion of arginine–vasopressin (AVP) and oxytocin (OXT) and thereby related physiological and behavioral functions, potentially leading to disorders in exposed subjects. The primary aim of this study was to demonstrate effects of chronic exposure to a low dose of an orally administered chlorobenzene mixture on anxiety-related and aggressive behavior mediated largely by AVP and OXT. Chlorobenzenes were applied to model ambient mixtures of endocrine disruptors. Adult, male Wistar rats were exposed daily to 0.1 μg/kg of 1,2,4trichlorobenzene and hexachlorobenzene via a stomach tube for 30, 60 or 90 days, after which anxiety-related and aggressive behavioral elements were examined in open-field, elevated plus maze and resident–intruder tests. The plasma levels of AVP, OXT and adrenocorticotrophic hormone at the endpoints were measured by radioimmunoassay or immunochemiluminescence assay. The levels of basal and serotonin- or norepinephrinestimulated AVP and OXT secretion in pituicyte cultures prepared from the posterior lobe of the pituitaries were also measured. The hormone levels proved to be increased to extents depending on the duration of exposure to the chlorobenzenes. Several anxiety-related and aggressive behavioral elements were also enhanced following chlorobenzene exposure, while certain explorative and locomotive elements of the animals were decreased. As both physiological and behavioral elements were modulated by chronic, subtoxic doses of chlorobenzenes, it is concluded that doses of such environmental pollutants low enough to fall outside the range of legal regulation may pose potential risks of anxiogenic and/or aggressive consequences in exposed subjects, including humans. © 2011 Elsevier Inc. All rights reserved.

1. Introduction

Abbreviations: 5-HT, serotonin (5-hydroxytryptamine); AC, absolute control; ACTH, adrenocorticotrophic hormone; AVP, arginine-vasopressin; ClB, chlorobenzene mixture applied for exposure in our experiments; HCB, hexachlorobenzene; NE, norepinephrine; OXT, oxytocin; POP/EDCs, persistent organic pollutants with endocrine disruptor potential; RIA, radioimmunoassay; SC, stress control; TCB, 1,2,4-trichlorobenzene. ⁎ Corresponding author. Tel.: + 36 62 545212; fax: + 36 62 545211. E-mail addresses: [email protected] (Z. Valkusz), [email protected] (G. Nagyéri), [email protected] (M. Radács), [email protected] (T. Ocskó), [email protected] (P. Hausinger), [email protected] (M. László), [email protected] (F.A. László), [email protected] (A. Juhász), [email protected] (J. Julesz), [email protected] (R. Pálföldi), galfi@jgypk.u-szeged.hu (M. Gálfi). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.03.016

Humans and wildlife may be exposed to stable, bioaccumulative, toxic pollutants [1], which may disseminate in the tissues and across the blood–brain barrier and the membranes of cells. Many such agents (persistent organic pollutants with endocrine disruptor potential, hereafter POP/EDCs) are proven or suspected to be able to interfere with central and/or peripheral endocrine elements [2]. Via endocrine targets in brain, POP/EDCs, acting alone or in ambient mixtures, may influence behavior through direct action on hormones [3,4]. The brain arginine–vasopressin (AVP) and oxytocin (OXT) have roles in the maintenance of homeostasis from behavioral and physiological aspects [5,6], and are necessary for adaptation to stress [6,7]. Even when exposed to stressors, homeostasis may be maintained via behavior, including anxiety and aggression, both mediated

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largely by AVP and OXT [6,8]. Anxiety enhances the capability and motivation of the individual to cope with stress and to attempt to deal with upcoming negative events [8,9]. Aggression, including intermale aggression [10], promotes better access to resources [8,11] and needed for effective communication within a society [8]. Behavior depends on secreted mediators. Brain AVP and OXT are expressed mainly by hypothalamic neurosecretory cells. The AVP and OXT-ergic peripheral system encompasses well-described hormonal effects resulting from secretion from axon terminals in the neurohypophysis. The central system includes the sites of expression in the brain, where functional (i.e. behavioral) consequences are usually partitioned from the peripheral ones by the limited permeability of the blood–brain barrier to AVP and OXT. However, the evidence indicates that, depending on stressors and stress conditions, centrally released neuropeptides, or even AVP and OXT secreted by hypothalamic magnocellular neurons, may affect the stress-induced activity of the hypothalamic–pituitary–adrenal axis [7]. The expressions of AVP and OXT at different brain sites are likewise modulated by various intrinsic regulators and extrinsic factors [6]. Serotonin (5-HT) and norepinephrine (NE), for instance, are important for both the production and secretion. These regulators (monoamines, adrenocorticotrophic hormone (ACTH), steroid hormones, etc.) may be implemented in anxiety and/or aggression mediated by secreted AVP and OXT [6,8]. If the amounts of secreted AVP and OXT are modulated for any reason within the specific brain areas responsible for the development of anxiety and/or aggression, behavioral consequences, psychiatric disorders may be manifested. It may be postulated that POP/EDCs that reach AVP- and OXTrelated brain areas may affect AVP and OXT secretion and thereby related functions. This can occur through action on central peptidergic cells, giving rise to changed behavior (acting in areas of central release) and/or physiological alterations (acting in areas of peripheral release, i.e. within the levels of the hypothalamo-neurohypophyseal system). Furthermore, theoretically all AVP and OXT-ergic and their support cells in brain may be the targets of POP/EDCs. However, despite the prevalence of AVP- and OXT-ergic elements, insufficient data are available on the effects of most POP/EDCs on the AVP- and OXT-related elements, the associated behavioral traits or functions. To date, POP/EDCs have been found in most tissues of exposed subjects [12], and there is increasing evidence that, by acting additively or synergistically, POP/EDCs may be biologically active even at extremely low doses [13]. The chlorobenzenes, and particularly hexachlorobenzene (HCB), have often been used as model agents [14] to investigate POP/EDCs. Although the use of HCB and many chlorobenzenes has been banned or controlled, they are still present environmentally [15–17]. Exposure may occur through inhalation, ingestion, dermal contact, placental or lactation-mediated transfer [17,18]. HCB accumulates in lipid-rich tissues such as endocrine glands [19], it is capable of affecting neural and endocrine elements and behavior [18], and it has been reported to be able to disturb parathyroid, thyroid or steroid [18] elements. Many frequent, active intermediate or by-product chlorobenzenes, such as 1,2,4-trichlorobenzene (TCB), have been demonstrated to be able to disturb thyroid functions [20]. Chlorobenzenes, alone or in mixtures, have been investigated mainly with regard to their physiological effects, though possible behavioral consequences could also influence homeostasis [16–18]. Most of the reported data on neurobehavioral effects relate to HCB, but they tend to emphasize either neurotoxic consequences or changes in cognitive behavior following exposure to at least mg or higher doses [18], i.e. doses higher than those usually found in the environment. Similarly, comparatively few reports have focused on emotional or social behavior and their possible underlying AVP-, OXTor other, e.g. 5-HT-related mechanisms. HCB contamination can result not only in neurotoxicological symptoms [21], but also in neuropsychiatric signs such as an increased frequency of schizophrenia and

hypochondria in patients with porphyria turcica [22]. Chronic dietary exposure of minks and European ferrets to major doses of HCB led to elevated aggressiveness and changes in the levels of monoaminergic regulators in various central nervous areas [23]. Chronic, but still discrete (1 μg/kg/day) exposure of Wistar rats to a mixture of such chlorobenzenes for 60 or 90 days led to elevated plasma levels of AVP and OXT, possibly due to modulated peripheral neuropeptide secretion, findings presumed to be related to the anxiogenic changes observed in behavioral tests [Nagyeri et al., submitted for publication]. Accordingly, in the present study we primarily set out to explore the changes in anxiety-related and intermale aggressive behavior of rats in response to chronic exposure to extremely low doses of chlorobenzenes, with the aim of drawing attention to the long-term, additive and harmful effects of subtoxic exposure to such agents, as environmental pollutants acting in ambient mixtures. We additionally initiated investigations of the possible changes in the secretion of AVP and OXT. Neurohypophyseal cell pituicytes were cultured from experimental rats and used as model to study neuropeptide secretion. 2. Materials and methods 2.1. Chemicals, materials Chemicals and cell culture materials were obtained from SigmaAldrich (St. Louis, MO), Invitrogen Corporation (Carlsbad, CA) or BDBiosciences (San Jose, CA), unless otherwise indicated. All chemicals were of analytical grade. The tissue culture medium was Dulbecco's Modified Essential Medium supplemented with 20% fetal calf serum and conventional components, as described earlier [24]. 2.2. Animals Medically certified male Wistar (Charles River, Isaszeg, Hungary) rats originating from different litters and weighing 300–350 g, aged 6–8 weeks at the beginning of the experiments, were used. After arrival, the animals were housed individually in standard plastic cages, assigned to various experimental groups (subsection 2.3 and Fig. 1) and kept under a controlled temperature of 22 ± 2 °C and relative air humidity of 55–65%. The animals lived under an automated, 12-h dark – 12-h light system (lights on at 6:00 a.m.) in the animal house of the Department of Physiology, Anatomy and Neuroscience at the University of Szeged. Smaller, unrelated Wistar male rats weighing 200–250 g, aged 4–6 weeks at the beginning,

Fig. 1. Experimental setup. The bold gray time axis denotes the duration of the study in days: the solid part indicates the exposure and the dashed part the days after exposure. ClB: groups exposed to chlorobenzenes for the specified numbers of days; SC: stress controls. Following open-field (OF) tests on day 1, elevated plus maze (EPM) tests on day 2, and resident–intruder (RI) tests on day 3, the rats were killed immediately after the RI tests and various in vitro procedures were performed: preparation of neurohypophyseal pituicyte cultures from day 3 to day 16–17 and monoamine incubation on day 16 or day 17; determination of AVP, OXT and ACTH levels of plasma collected on day 3 and neurohypophysis cell culture supernatant samples prepared on day 16 or 17.

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maintained in another room under the same conditions as mentioned above, were used as intruder animals for the resident–intruder tests (subsection 2.4.3). Laboratory rodent chow (CRLT/N, Charles River, Gödöllő, Hungary) and tap water were available ad libitum. This diet contained 86% dry material, 19% crude protein, 17% digestible protein, 4.5% crude fat, 6% crude fiber, 6% crude ash, 40% non-protein nitrogen material, 0.8% calcium, 11000 IU/kg vitamin A, 600 IU/kg vitamin D3, plus the amino acids lysine, methionine and cysteine. The tap water was analyzed chemically by the Szeged Water Company. The acclimation of the animals lasted for 2 weeks. The care and the procedures were carried out in strict accordance with European Communities Council Directive 86/609/EEC. 2.3. Exposure, experimental groups and protocol A 1:1 mixture of 1,2,4-trichlorobenzene (CAS registry number: 120-82-1) and hexachlorobenzene (CAS registry number: 118-74-1) in 1 mL of 0.015% aqueous ethanol in tap water was administered daily in a dose of 0.1 μg/kg via a stomach tube. This mixture is referred to below as ClB. The chlorobenzenes used were arbitrarily chosen, as a model of ambient mixture of POP/EDCs. The dosage and duration of ClB exposure were selected on the basis of earlier non-behavioral studies, and regard to oral exposure to doses of HCB or TCB combined with the lowest observed effects described in literature [16–18]. The rats were exposed to ClB for 30 (n = 10, ClB-30 group), 60 (n = 10, ClB-60 group) or 90 (n = 10, ClB-90 group) days, or to tube insertion only (stress controls, SC) for 30 (n = 5, SC-30 group), 60 (n = 5, SC-60 group) or 90 (n = 5, SC-90 group) days likewise. Absolute controls (referred to below as controls or AC group) were unexposed (n = 10). The protocol is depicted in Fig. 1. Following ClB or stress exposure, anxiety-related, locomotive and explorative behavioral elements were examined in open-field and elevated plus maze tests, and the intermale aggression was studied via the neutral cage paradigm of resident–intruder tests. After the final behavioral testing, each animal was immediately killed by rapid decapitation always at approximately the same time of the day, and its trunk blood was collected to measure the plasma levels of AVP, OXT and ACTH (subsection 2.7.2) and of alanine transaminase, aspartate aminotransferase and gammaglutamyl transpeptidase (subsection 2.8). The brain, liver, kidneys and pituitaries were carefully removed. Neurohypophyseal cell pituicyte cultures were prepared from the posterior part of the excised pituitaries (subsection 2.5), and the levels of basal and 5-HT- and NE-mediated AVP and OXT secretion of the 13–14-day-old, confluent neurohypophysis cultures were determined (subsection 2.6). The AVP and OXT contents of the supernatants of the neurohypophysis cultures were assayed (subsection 2.7.1). Basic toxicological and morphological examinations were performed on the animals or their organs throughout the experiments (subsection 2.8). 2.4. Behavioral experiments Animals participated in each test only once. All tests were performed in the early hours of the dark (active) phase. The animals were transferred to the test room under identical conditions (subsection 2.2), and allowed to become habituated for 1 h. The subjects were removed from the cages in a random sequence for testing. The test apparatus was always cleaned with ethanol before each trial in order to eliminate odorous cues. Behavior was videotaped by a camera mounted on the ceiling directly above the apparatus and recorded with Ethovision software (v2.3, Noldus Information Technology, Wageningen, The Netherlands). Each test was reanalyzed by an observer unaware of the nature of the experiments. 2.4.1. Open-field tests Open field tests [25] were performed to observe behavioral elements relating to anxiety or locomotion and exploration. The

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subject was placed at the center of a circular (diameter 80 cm, height 45 cm), plastic, black-painted arena open at the top, and the percentages of time spent in the center and the periphery, the total distance moved, the mean velocity, and the total durations and numbers of rearing, grooming, sniffing, freezing and defecation episodes were measured. Each test lasted for 5 min. 2.4.2. Elevated plus maze tests Elevated plus maze tests [26] were likewise performed to determine the consequences of exposure on anxiety-related behavioral elements, locomotion and exploration. The elevated (50 cm from the floor) apparatus (brown-painted wood) consisted of two opposing open arms (50 × 8 cm), two opposing closed (50 × 8 × 15 cm) arms and a central area (8 × 8 cm). The floor under the apparatus was covered with dark cloth to reduce height cues. Animals were initially placed at the center, always facing the same open arm, and the times spent in the open and closed arms, the “preferred site” (which was calculated with a ratio of the total duration % in open arms and the total duration % in whole apparatus), the numbers of entries into the various zones, and the total durations and numbers of head-dipping, rearing, grooming, sniffing and freezing episodes were measured. Each test lasted for 3 min. 2.4.3. Resident–intruder tests Resident–intruder tests with the neutral cage paradigm were applied to determine possible changes in intermale aggressive behavior following exposure to ClB. The resident animal was placed at the center of an arena for habituation, and was left to explore the area for 5 min. The arena was a neutral cage strewn with unused, clean wooden chips and covered with a transparent, plastic lid. The parameters relating to locomotion, exploration and self-care, as measured in the open-field tests (subsection 2.4.1), were recorded throughout the habituation and the subsequent resident–intruder test. After habituation, an intruder animal was introduced to the resident and various offensive (total durations and numbers of aggressive grooming episodes, lateral threats, menacing postures, chasing and biting attacks), defensive (total durations and numbers of defensive upright posture and immobility) and social (naso-nasal and naso-genital contacts) behavioral elements of the residents were recorded, as described earlier [27]. The behavior of the intruder animals was not analyzed. Each test lasted for 5 min. 2.5. Cell culture techniques Neurohypophyseal cell pituicyte cultures were prepared from excised, sterile pituitaries as described earlier [24]. The procedures with the glial cells and the pituicytes were as published earlier [28–30]. The cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium on the cultured pituicytes was changed daily before the incubation procedures (subsection 2.6). 2.6. Incubation procedures with monoamines The conditions and the dose/time relationships regarding 5-HT, NE, AVP and OXT secretion were determined previously [31–33]. Confluent (13–14-day-old) monolayers were always used for incubation procedures. Before any manipulations, the medium was changed and incubation was performed for 2 h, after which supernatant aliquots were collected (designated NH 1, no treatment) and frozen at −70 °C until radioimmunoassay (RIA) was performed. For monoamine-mediated secretion determinations, neurohypophysis cultures were incubated with 10− 6 M 5-HT (NH 2) or NE (NH 3) for 1 h. The dose and duration of treatment were chosen on the basis of previous findings [31,32]. Following incubation, supernatant aliquots were collected and kept refrigerated at − 70 °C until hormone measurements (subsection 2.7).

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2.7. Hormone assay

3. Results

2.7.1. Determination of AVP and OXT secretion of neurohypophysis cultures AVP and OXT radioimmunoassays were performed as reported earlier [24]. Synthetic AVP (Organon, Oss, The Netherlands) and OXT (Richter, Budapest, Hungary) were used as reference preparations for antibody production and radiolabeling procedures. Antibody was generated against either AVP-(e-aminocaproic acid)-thyroglobulin conjugate in sheep, or OXT-(e-aminocaproic acid)–thyroglobulin conjugate in New Zealand rabbits. The immunization in both procedures consisted of injections every 2 weeks for 12 weeks. The final antibody dilution in the assay tube was 1:350,000 for AVP and 1:70,000 for OXT. In the AVP assay, the cross-reactions were 23.3% with lysine-vasopressin, 0.01% with OXT and 0.03% with vasotocin, while in the OXT assay, the cross-reactions were 92.7% with oxypressin, 0.11% with vasotocin and 0.05% with AVP, lysinevasopressin or MIF (Pro-Leu-Gly-NH2). In the AVP assay, the intraand interassay coefficients of variation proved to be 13.3% and 16.3%, respectively, while in the OXT assay, they were less than 15%. 125Ilabeling of AVP and OXT was performed by the chloramine-T method, and reverse-phase chromatography was used for purification of these tracers. Neurohypophyseal supernatant samples directly and blood samples following preparation processes (subsection 2.7.2) were assayed by RIA [24]. The standard curves covered the range 1.0 to 128 pg per assay tube. The sensitivity of both assays was 1 pg/tube. The total protein contents of samples were measured with the BCA Protein Assay Kit (catalog number 23225, Fisher Scientific, Chicago, IL).

3.1. Stress controls versus untreated animals

2.7.2. Determination of plasma AVP, OXT and ACTH Blood samples were collected in chilled, Na2EDTA-coated polystyrene tubes and centrifuged immediately, and plasma aliquots were separated and stored at − 70 °C until measurements. Following extraction on Amprep C8 minicolumns (Y2-VW-RPN1902; Amersham, UK) with a recovery of ≥95%, plasma AVP and OXT levels were assayed by RIA (subsection 2.7.1). Plasma ACTH levels were measured by immunochemiluminescence assay with an Immulite 2000 apparatus (Siemens Healthcare Diagnostic, Deerfield, IL) and DPC kit (L2KAC-02; Euro/DPC Ltd, Glyn Rhonwy, UK). 2.8. Determination of toxicity of applied exposures Alanine transaminase, aspartate aminotransferase and gammaglutamyl transpeptidase, enzymes often used as markers of liver damage induced by environmental agents, including HCB exposure [34], were measured by conventional methods. The animals were weighed regularly throughout the experiments. After sacrifice of the animals, the brain, liver, spleen and kidneys were removed and weighed. Signs indicative of HCB or TCB toxicity, e.g. alopecia, skin thickening, scarring and erythema [18], were monitored, and general morphologic examinations with conventional histological stains such as hematoxylin–eosin staining were performed on specimens prepared from excised organs. 2.9. Statistical analysis The program Statistica 9.0 (Statsoft, Tulsa, OK) was used for data analyses. Data were processed by two-way analysis of variance, with treatment (ClB or SC) and duration of treatment (30, 60 or 90 days) as independent factors. Groups were compared by Fisher's LSD post hoc test, and the ClB and SC groups were compared with the AC group by the Dunnett post hoc test. The results are presented as means ± standard error of mean (SEM). Changes were generally considered statistically significant at p b 0.05.

No statistical differences in any of the measured parameters were found between the AC and SC groups. Significant differences between the ClB-exposed and AC groups (subsection 2.9) are indicated in Figs. 2-6. 3.2. Behavioral changes following ClB exposure The results of open-field, elevated plus maze and resident– intruder tests are presented in Figs. 2 and 3. 3.2.1. Behavior in open-field tests The percentage of time spent in the periphery (Fig. 2A1, upper part) was increased statistically in the ClB-60 and ClB-90 groups, as was the number of freezing episodes in the ClB-90 group (Fig. 2A1, lower part). The total duration (Fig. 2B2, upper part) and number (Fig. 2B1, upper part) of rearings were reduced significantly in the ClB-30, ClB-60 and ClB-90 groups. The total distance moved (Fig. 2C1, upper part) and the mean velocity (Fig. 2C2, upper part) were mainly significantly reduced in the ClB-30, ClB-60 and ClB-90 groups. 3.2.2. Behavior observed in elevated plus maze tests The preferred site (i.e. the ratio of the time spent in the open arms and the time spent in the whole apparatus) was decreased significantly in the ClB-60 group (Fig. 2A2). The total duration of rearings (Fig. 2B2, lower part) was not altered significantly, but the number of rearings (Fig. 2B1, lower part) in the closed arms was modulated in all exposed groups. The total distance moved (Fig. 2C1, lower part) and the mean velocity (Fig. 2C2, lower part) were decreased significantly by ClB treatment for 30, 60 or 90 days. 3.2.3. Behavior in resident–intruder tests As concerns the offensive parameters, significant increases were observed in the number of lateral threats (Fig. 3A, upper part), the number of menacing postures (Fig. 3B, lower part) and the incidence of chasing (Fig. 3B, upper part) in the ClB-90 group. No statistical differences were found in the incidence of attack biting (Fig. 3A, lower part) or other behavioral elements (subsections 2.4.1–2.4.3) in the openfield, elevated plus maze or resident–intruder tests (data not shown). 3.3. AVP and OXT secretion of neurohypophyseal cell cultures After incubation with the monoamines (NH 1–3; subsection 2.6), the AVP and OXT contents of the supernatant aliquots from neurohypophyseal cultures were assayed by RIA (Fig. 4). 3.3.1. NH 1 — basal AVP and OXT levels The basal secretion of both AVP and OXT was slightly modulated in the ClB-30 group, but statistically significantly elevated in the ClB-60 group and even more so in the ClB-90 group (Fig. 4A). 3.3.2. NH 2 — 5-HT-stimulated AVP and OXT levels Statistical differences were found between the groups as concerns AVP and OXT secretion. As reported earlier [31], the basal levels of both AVP and OXT secretion of the neurohypophyseal cultures prepared from healthy, unexposed rats were significantly increased following treatment with 10− 6 M 5-HT, as were the levels of 5-HTinduced AVP secretion in all three ClB groups, and of 5-HT-induced OXT secretion in the ClB-60 and ClB-90 groups (Fig. 4B). 3.3.3. NH 3 — NE-stimulated AVP and OXT levels Similarly to NH 2, Statistical differences were found between the exposed groups and controls in AVP and OXT secretion. As reported

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Fig. 2. A. Anxiety-related behavioral consequences of exposure to ClB, measured in open-field and elevated plus maze tests. Means ± SEM, n = 10/group. A1: Anxiety-related elements: time spent in the periphery (upper part; ordered to left y axis) and freezing (lower part; ordered to right y axis), both measured in open-field tests. A2: Anxiety-related element: preferred site, measured in elevated plus maze tests. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001. B. Explorative behavioral consequences of exposure to ClB, measured in open-field and elevated plus maze tests. Means ± SEM, n = 10/group. B1: Explorative element: number of rearing episodes measured in open-field (upper part) and elevated plus maze (lower part) tests. B2: Explorative element: duration of rearing episodes measured in open-field (upper part) and elevated plus maze (lower part) tests. The explorative elements in the elevated plus maze test were measured in the closed arms. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001. C. Locomotive behavioral consequences of exposure to ClB, measured in open-field and elevated plus maze tests. Means ± SEM, n = 10/group. C1: Locomotive element: total distance moved, measured in open-field (upper part) and elevated plus maze (lower part) tests. C2: Locomotive element: average velocity measured in open-field (upper part) and elevated plus maze (lower part) tests. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001.

earlier [33,35], the basal levels of AVP and OXT secretion were significantly increased following treatment with 10− 6 M NE. The level of NE-induced AVP secretion was increased significantly in all three ClB groups, as was that of NE-induced OXT secretion in the ClB-60 and ClB-90 groups (Fig. 4C).

3.4. Plasma AVP, OXT and ACTH levels Following the rapid decapitation of the animals (Fig. 1), trunk blood was collected to determine circulating hormone levels. The plasma levels of AVP (Fig. 5A, lower part), OXT (Fig. 5A, upper part)

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Fig. 3. Behavioral consequences of exposure to ClB measured in resident–intruder tests. Means ± SEM, n = 10/group. A: Offensive elements: numbers of lateral threats (upper part; ordered to left y axis) and attack bitings (lower part; ordered to right y axis). B: Offensive elements: numbers of chasings (upper; part, ordered to left y axis) and menacing postures (lower part; ordered to right y axis). Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: ⁎⁎p b 0.01.

and ACTH (Fig. 5B) were significantly elevated in the ClB-60 and ClB90 groups. 3.5. Toxicity of ClB exposure The body and organ weight results did not reveal any significant differences between the experimental groups and the controls (data not shown). Nor were statistical differences detected in aspartate aminotransferase (Fig. 6, upper part), alanine transaminase (Fig. 6, middle part) or gamma-glutamyl transpeptidase (Fig. 6, lower part). No malformations or other overt signs (e.g. alopecia) of toxicity were observed. No structural differences were observed on slides prepared from excised brain, liver, spleen and kidney for standard microscopical examinations. 4. Discussion 4.1. Exposure to low doses of POP/EDC chlorobenzenes HCB and certain chlorobenzenes such as TCB are able to disturb central and/or peripheral endocrine targets [2,20], and can be neurologically or immunologically toxic [18]. HCB has known impacts on various aspects of behavior [18,36]. Their accumulation in soil, water, air, sediments and the food chain adds to the relevance of HCB [14,37], chlorobenzenes [17] and POP/EDCs [2]. Primarily the consequences of exposure to doses of POP/EDCs an order of magnitude higher than those usually found in the environment have been

Fig. 4. A. AVP and OXT secretion of neurohypophysis cultures — NH 1. Means ± SEM, n = 10 cultures/neurohypophysis treatment/experimental groups. All results are given in pg hormone/mg protein. AVP (lower part) and OXT (upper part) concentrations. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: ⁎p b 0.05, ⁎⁎⁎p b 0.001. B. AVP and OXT secretion of neurohypophysis cultures — NH 2. Means ± SEM, n = 10 cultures/ neurohypophysis treatment/experimental groups. All results are given in pg hormone/mg protein. AVP (lower part) and OXT (upper part) concentrations. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: (⁎⁎⁎p b 0.001). C. AVP and OXT secretion of neurohypophysis cultures — NH 3. Means ± SEM, n = 10 cultures/neurohypophysis treatment/experimental groups. All results are given in pg hormone/mg protein. AVP (lower part) and OXT (upper part) concentrations. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significant differences: ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001.

studied to date, though their prevalence in the environment and their potential to affect wildlife and human populations adversely should receive more recognition among scientists, policy-makers and the general public alike. Low amounts of POP/EDCs often result in no overt

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ination on the body or excised organs. The results indicate that the dose (0.1 μg/kg/day) and duration (30, 60 or 90 days) of exposure to ClB applied in this study must be considered subtoxic. 4.2. Regulation mechanisms in AVP and OXT secretion

Fig. 5. Plasma levels of AVP, OXT and ACTH following exposure to ClB. Means ± SEM, n = 10/group. Hormones measured in trunk blood collected at endpoints of experiments. Results are given in pg hormone/mL plasma. A: AVP (lower part) and OXT (upper part) concentrations. B: ACTH concentrations. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers. Significance: ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001.

symptoms or effects and therefore neglected, whereas long-term exposure to discrete doses may involve potential health risks [38]. Our study included basic toxicological routines to decide whether ClB was subtoxic. Following exposure, the body and organ weights did not reveal significant differences between the ClB-exposed groups and the stress- or absolute controls (data not shown); nor did the plasma levels of liver transferases (Fig. 6). We were unsuccessful in demonstrating published signs [17,18] of toxic HCB or TCB contam-

Independently or in interaction with other regulators, AVP and OXT are involved in the manifestation of many behavioral forms, including anxiety and aggression [6,8]. The interactions between neuropeptides and 5-HT- or NE-related neurogenic systems have been widely discussed at different levels of the secretion and production or transmission within the intra- and extrahypothalamic superior nuclei and brain areas, but less so at the level of the neurohypophysis, a lower regulating area of hypothalamo-neurohypophyseal AVP and OXT release. In the hypothalamus, the paraventricular and supraoptic nucleus receive both 5-HT- and NE-ergic innervations and house the related receptors [39]. Such inputs are involved in the release and synthesis of AVP and OXT when increased hormone levels are necessary. NE increases AVP and OXT expression in both nuclei, while 5-HT stimulates AVP and OXT expression in paraventricular nucleus, but only that of OXT in the supraoptic nucleus [40,41]. Mutual roles of AVP and 5-HT [42] and/or NE [43] have been described in emotional or social behavior and related disorders, these roles mainly involving 5-HT receptors in brain areas related to AVP and OXT release or production [44,45]. The participation of 5-HT receptors in the mediation of stress-induced AVP and OXT secretion [46] has also been identified. The NE-ergic system enhances the central and peripheral release of neuropeptides, especially via participation of the histaminergic neurons [47]. However, NE may inhibit AVP secretion [48,49]. This contradiction has been explained in that adrenoceptors may be distributed differentially on the surface of the AVP-ergic cells, allowing different adrenergic inputs to be excitatory or inhibitory [50]. NE may further regulate the peripheral release of OXT. NE-ergic varicosities in contact with AVP and OXT in magnocellular neurons have been under basal conditions, but the density of these varicosities apposed to somata of the OXT neurons increases significantly in the paraventricular and supraoptic nuclei during lactation [51]. Depletion of hypothalamic NE decreases the peripheral OXT release in response to footshock, the peripheral administration of cholecystokinin or hypertonic saline or suckling during lactation [52]. Pituicytes may also be involved in AVP and OXT secretion and production. AVP and OXT were long considered only to be stored and not produced in the neurohypophysis [53], but our group [24] and others have clearly demonstrated that the neurohypophysis can also be the site of AVP and OXT expression. Moreover, pituicytes are sensitive to osmotic changes [54,55], and the activation of AVP expression may be due to the osmosensitivity of neurohypophyseal cells [56,57]. Receptors and innervations relating to the monoaminergic regulators are to be found in several areas of the hypothalamo-neurohypophyseal system [35,58]. In vitro neurohypophyseal cultures can be used to investigate basal and monoamine-mediated AVP and OXT expression because of the possibility of neuropeptide release and synthesis [24] and the presence of receptors and mediated functions of 5-HT [31] and NE [32,33,35]. We earlier have postulated that, under in vivo conditions, monoaminergic involvement may appear at the level of the neurohypophysis, independently of the hypothalamus. Our earlier findings suggest similar roles of monoaminergic regulators at higher levels (in intra- and extrahypothalamic superior neurons), which could be affected in the development of AVP- and OXT-mediated behavior. 4.3. Influence of exposure to ClB on AVP and OXT secretion in pituicyte model

Fig. 6. Changes in plasma levels of liver damage markers following exposure to ClB. Means ± SEM, n = 10/group. Markers were measured in trunk blood collected at endpoints of experiments. The results are given in IU enzyme/L plasma. Absolute controls: day 0. Stress controls: dashed, light-gray lines and markers. ClB-treated animals: solid, dark-gray lines and markers.

To investigate ClB-induced changes in basal and monoaminemediated AVP and OXT secretion, incubation with monoaminergic receptor-specific mediators (NH 1–3; subsection 2.6) was performed

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(Fig. 4). We reported earlier that 5-HT and NE (and others, e.g. dopamine or histamine) applied as receptor agonists enhanced both the AVP [31,32] and the OXT [31,33] secretion of pituicytes in vitro. These effects could be prevented by the preliminary addition of receptor antagonists; the receptors acting in the AVP and OXT secretion of neurohypophyseal cultures were therefore identified [31–33]. Similarly as when a higher (1 μg/kg/day) dose of ClB was used as environmental exposure [Nagyéri et al., submitted for publication], exposure to a 0.1 μg/kg/day dose of ClB altered even the basal neuropeptide secretion; the effects were found to vary to extents depending on the duration. In cultured neurohypophyseal cells from ClB-exposed animals, the secretion of AVP and OXT was likewise elevated by monoaminergic agonists, but in a modified manner, probably due to the impact of ClB. We presumed that the basal and monoamine-mediated secretion of AVP and OXT was modulated under in vitro conditions by the ClB exposure, and hypothesized that such modulation can also occur at the level of the neurohypophysis under in vivo conditions. Most POP/EDCs, including HCB, can reach the brain [22] and accumulate in neurons or glial cells, for instance. In the long term, the chlorobenzenes can attain behaviorrelated (and neuropeptide-expressing) areas and the areas of the hypothalamo-neurohypophyseal system, including the pituicytes in the neurohypophysis. As dioxin-like agents, chlorobenzenes may induce many intracellular processes [59,60]. For instance, HCB has been reported to affect phospholipid patterns in the brain, altering the functional efficacy of neuronal membranes, and resulting in changes in membrane properties and consequently in the activity of embedded proteins such as receptors, ion-channels, etc. [61]. It may be suspected that the changes observed in vitro in both basal and monoamine-mediated neuropeptide secretion could also appear at various levels of AVP and OXT secretion. The differences may be caused by modification of membrane receptors and their sensitivities, or by changes in transmitter amounts or other HCB-related mechanisms. Our present assumptions appear to be in accord with the earlier findings that the chronic administration of dietary HCB to minks and European ferrets led not only to abnormally high aggressiveness and hyperexcitability, but also to elevated 5-HT and NE levels in the hypothalamus and other brain areas [23]. 4.4. Effects of exposure to ClB on stress-related functions In the present work, exposure for 30 days did not change the plasma AVP and OXT levels appreciably, whereas exposure for 60 or 90 days increased the levels significantly (Fig. 5A), possibly as a consequence of modulated peripheral AVP and OXT secretion. Our data tend to correlate with the increased plasma AVP levels in patients with various anxiety disorders [62–64]. However, our findings concerning the plasma OXT levels seem to disagree with earlier (usually negative) correlations between OXT levels and anxiety scores [65]. The neurohypophyseal OXT, which is primarily synthesized in hypothalamic magnocellular nuclei, is released during stress and is believed to be anxiolytic. The plasma OXT may be increased by certain stressors, and the OXT released mainly from the paraventricular nucleus may play a role in physiological responses to stress [7]. In one study [66], when male rats were stressed by shaking, enhanced OXT secretion was detected in the paraventricular nucleus, with an accompanying elevation in the plasma OXT level. To the best of our knowledge, the effects of chlorobenzene-induced stress on OXTrelated mechanisms have not been investigated previously. We can state only that our SC groups did not display increased OXT (or AVP) levels; the ClB stress may have induced peripheral OXT release. Few data are available on the correlation between the plasma AVP (or OXT) level and aggressive behavior. Rather than neuropeptides, the plasma levels of 5-HT, testosterone or glucocorticoids are mainly mentioned as correlating regulators of aggressive behavior. However,

in the cerebrospinal fluid, the concentration of AVP (and OXT and other regulators) usually correlated dose-dependently with aggression [8]. Significantly increased plasma levels of ACTH (Fig. 5B) were found following exposure for 60 or 90 days. AVP and corticotropinreleasing hormone released into the portal circulation at the median eminence or even the AVP and OXT released within the neurohypophysis into the short portal vessels are known to affect ACTH secretion in the adenohypophysis, which triggers the adrenal release of glucocorticoids and facilitates physiological and behavioral adaptation to stressors at both peripheral and brain levels [7]. 4.5. Effects of exposure to ClB on behavioral functions After ClB exposure, anxiety-related (Fig. 2A), explorative (Fig. 2B) and locomotive behavioral elements (Fig. 2B) were detected in openfield and elevated plus maze tests [25]. Both tests can be used for the examination of anxiety-related behavior [25,26]. Elements of intermale aggression were also observed in resident–intruder tests by using the neutral cage paradigm (Fig. 3). Similarly as reported previously [Nagyéri et al., submitted for publication], the anxietyrelated behavior was altered following exposure to ClB for 60 or 90 days, and discrete alterations in locomotive and/or explorative behavior were also detected. Besides the changes in anxiety-related elements, the total distance moved and the mean velocity of the movement were decreased in open-field tests following 30, 60 or 90-day ClB treatment. The numbers of rearings (as explorative elements) were also significantly lower in the ClB-exposed rats. These elements can be used as markers of locomotor activity and explorative behavior. The level of anxiety is reflected in open-field tests by the changes in the time spent in the periphery and in explorative activity, and in elevated plus maze tests by the differences in the proportions of time spent in the open or closed arms and in explorative behavior [25]. Therefore, our results suggest that a 0.1 μg/kg/day dose of ClB for 90 days may be considered to have a discrete anxiogenic environmental impact. Fewer consequences of ClB exposure were observed as concerns aggressive behavior. The lateral threats, chasing and a menacing posture seemed to be affected significantly. However, other offensive, defensive or social behavioral elements did not differ statistically. Thus, the resident–intruder tests indicated that the subtoxic doses of ClB applied had only minor effects on intermale aggression. Higher doses of orally applied ClB exposures earlier provoked higher aggressiveness observed by our colleagues (unpublished), but these findings were not corroborated by behavioral tests and those studies were not designed to observe behavioral changes induced by chlorobenzenes. It is generally accepted that POP/EDCs may alter a wide variety of behavior, including sexual and reproductive behavior, communication, dominance, aggression and cognitive elements such as attention, learning and memory. The mechanisms are still often unclear, or the attempts to explain them involve known target hormones (usually steroids and/or thyroids) at the level of synthesis, storage, release, transport, clearance, receptor, or receptor recognition) within the relevant brain areas [3]. Unfortunately, few data are available on the effects of POP/EDCs as regards anxiety or other neuropeptide-mediated behavior; this also holds for chlorobenzenes. The published papers suggest that the long-term presence of POP/EDC agents (with features similar to chlorobenzenes) in considerable doses often leads to appreciable anxiogenic effects, as in the case of bisphenol A [67]. Positive, negative and no effects relating to aggression have all been described, depending on the type of POP/EDC, the exposure, the exposed subjects, and the experimental conditions [3]. 4.6. Conclusions and future directions In conclusion, it may be postulated that chronic exposure to extremely low doses of endocrine disruptor chlorobenzenes may

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influence certain AVP- and OXT-related behavioral traits and underlying neuroendocrine systems. Our findings may relate to disturbances in AVP and OXT secretion. Chlorobenzenes may act as discrete anxiogenous factors and may also have the potential to influence various behavioral elements of aggression. Consequently, as frequent environmental pollutants, these chemicals may pose potential risks in the etiology of psychiatric disorders with symptoms of abnormal anxiety and/or aggressiveness. These chemical impacts are rather general, since individuals may easily come into contact with contaminated foodstuffs or potable water, for example. Subtoxic doses of POP/EDCs or their mixtures often do not appear to be known to be harmful to health. However, policy-makers, law-makers and the general public should perhaps pay more attention to the danger of exposure to low doses of POP/EDCs. This study should be only the first step in a longer series of experiments. We have shown that exposure to low doses of POP/EDCs can induce discrete alterations in behavior. The consequences of ClBinduced changes in behavior, however, are still unclear. At present we do not fully understand the mechanisms of the findings. We may only suggest the involvement of known cellular effects of HCB. To clarify the mechanisms, further, more comprehensive analyses are essential. We plan to examine AVP and OXT secretion in different superior brain areas (among them the magnocellular and other nuclei) directly associated with AVP and OXT-related behavior, including anxiety and aggression. More sophisticated investigations will be performed in connection with subjects exposed to ClB, e.g. in vivo microdialysis procedures, immunohistochemistry, in situ hybridization processes and chromatographic methods for the measurement of secreted AVP and OXT or accumulated ClB.

Acknowledgments The authors are especially grateful to their colleagues at the Department of Medical Chemistry for help and advice. They acknowledge financial support from grants Hungarian National Development Agency grants TAMOP 4.2.2-08/1-2008-0007, TAMOP 4.2.2-08/1-2008-0006 and TAMOP 4.2.1/B-09/1/KONV-2010-0005.

References [1] Breivik K, Alcock R, Li YF, Bailey RE, Fiedler H, Pacyna JM. Primary sources of selected POPs: regional and global scale emission inventories. Environ Pollut 2004;128:3–16. [2] Colborn T, vom Saal FS, Soto AM. Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ Health Perspect 1993;101:378–84. [3] Zala SM, Penn DJ. Abnormal behaviours induced by chemical pollution: a review of the evidence and new challenges. Anim Behav 2004;68:649–64. [4] Vos JG, Dybing E, Greim HA, Ladefoged O, Lambre C, Tarazona JV, et al. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol 2000;30:71–133. [5] Ugrumov MV. Developing brain as an endocrine organ: a paradoxical reality. Neurochem Res 2010;35(6):837–50. [6] Landgraf R, Neumann ID. Vasopressin and oxytocin release within the brain: a dynamic concept of multiple and variable modes of neuropeptide communication. Front Neuroendocrinol 2004;25:150–76. [7] Engelmann M, Landgraf R, Wotjak CT. The hypothalamic-neurohypophysial system regulates the hypothalamic–pituitary–adrenal axis under stress: an old concept revisited. Front Neuroendocrinol 2004;25:132–49. [8] Neumann ID, Veenema AH, Beiderbeck DI. Aggression and anxiety: social context and neurobiological links. Front Behav Neurosci 2010;4:12. [9] Frank E, Landgraf R. The vasopressin system—from antidiuresis to psychopathology. Eur J Pharmacol 2008;583:226–42. [10] Blanchard RJ, Wall PM, Blanchard DC. Problems in the study of rodent aggression. Horm Behav 2003;44:161–70. [11] Caldwell HK, Lee HJ, Macbeth AH, Young III WS. Vasopressin: behavioral roles of an “original” neuropeptide. Prog Neurobiol 2008;84:1–24. [12] Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM, et al. Endocrine-disrupting chemicals: an endocrine society scientific statement. Endocr Rev 2009;30:293–342. [13] McKinlay R, Plant JA, Bell JN, Voulvoulis N. Endocrine disrupting pesticides: implications for risk assessment. Environ Int 2008;34:168–83.

429

[14] Barber JL, Sweetman AJ, van Wijk D, Jones KC. Hexachlorobenzene in the global environment: emissions, levels, distribution, trends and processes. Sci Total Environ 2005;349:1–44. [15] Bailey RE. Global hexachlorobenzene emissions. Chemosphere 2001;43:167–82. [16] California Public Health Goal. 1,2,4-trichlorobenzene in drinking water. Office of Environmental Health Hazard Assessment California Environmental Protection Agency; 1999. [17] IPCS. Chlorobenzenes other than hexachlorobenzene: environmental aspect. Concise International Chemical Assessment Document 60. Geneva: World Health Organization, International Programme on Chemical Safety; 2004. [18] ATSDR. Toxicological profile for hexachlorobenzene. Atlanta : Agency for Toxic Substances and Disease Registry; 2002. [19] Ingebrigtsen K, Nafstad I. Distribution and elimination of C-14-Labeled hexachlorobenzene after single oral-exposure in the male-rat. Acta Pharmacol Toxicol 1983;52:254–60. [20] den Besten C, Vet JJ, Besselink HT, Kiel GS, van Berkel BJ, Beems R, et al. The liver, kidney, and thyroid toxicity of chlorinated benzenes. Toxicol Appl Pharmacol 1991;111:69–81. [21] Evangelista deDuffard AM, Duffard R. Behavioral toxicology, risk assessment, and chlorinated hydrocarbons. Environ Health Perspect 1996;104:353–60. [22] Peters HA, Cripps DJ, Gocmen A, Erturk E, Bryan GT, Morris CR. Neurotoxicity of hexachlorobenzene-induced porphyria turcica. IARC Sci. Publ; 1986. p. 575–9. [23] Bleavins MR, Bursian SJ, Brewster JS, Aulerich RJ. Effects of dietary hexachlorobenzene exposure on regional brain biogenic amine concentrations in mink and European ferrets. J Toxicol Environ Health 1984;14:363–77. [24] Janaky T, Szabo P, Kele Z, Balaspiri L, Varga C, Galfi M, et al. Identification of oxytocin and vasopressin from neurohypophyseal cell culture. Rapid Commun Mass Spectrom 1998;12:1765–8. [25] Klivenyi P, Bende Z, Hartai Z, Penke Z, Nemeth H, Toldi J, et al. Behaviour changes in a transgenic model of Huntington's disease. Behav Brain Res 2006;169:137–41. [26] Biro E, Penke B, Telegdy G. Role of different neurotransmitter systems in the cholecystokinin octapeptide-induced anxiogenic response in rats. Neuropeptides 1997;31:281–5. [27] Mikics E, Kruk MR, Haller J. Genomic and non-genomic effects of glucocorticoids on aggressive behavior in male rats. Psychoneuroendocrinology 2004;29:618–35. [28] Galfi M, Janaky T, Toth R, Prohaszka G, Juhasz A, Varga C, et al. Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures. Regul Pept 2001;98:49–54. [29] Kennedy PG, Lisak RP, Raff MC. Cell type-specific markers for human glial and neuronal cells in culture. Lab Investig 1980;43:342–51. [30] Michlerstuke A, Bottenstein JE. Proliferation of glial-derived cells in defined media. J Neurosci Res 1982;7:215–28. [31] Galfi M, Radacs M, Juhasz A, Laszlo F, Molnar A, Laszlo FA. Serotonin-induced enhancement of vasopressin and oxytocin secretion in rat neurohypophyseal tissue culture. Regul Pept 2005;127:225–31. [32] Radacs M, Galfi M, Nagyeri G, Molnar AH, Varga C, Laszlo F, et al. Significance of the adrenergic system in the regulation of vasopressin secretion in rat neurohypophyseal tissue cultures. Regul Pept 2008;148:1–5. [33] Radacs M, Molnar AH, Laszlo FA, Varga C, Laszlo F, Galfi M. Inhibitory effect of galanin on adrenaline- and noradrenaline-induced increased oxytocin secretion in rat neurohypophyseal cell cultures. J Mol Neurosci 2010;42(1):59–66. [34] Adjarov D, Ivanov E, Keremidchiev D. Gamma-glutamyl transferase: a sensitive marker in experimental hexachlorobenzene intoxication. Toxicology 1982;23: 73–7. [35] Nagyeri G, Galfi M, Radacs M, Molnar AH, Laszlo F, Varga C, et al. Effects of galaninmonoaminergic interactions on vasopressin secretion in rat neurohypophyseal cell cultures. Regul Pept 2009;155:76–80. [36] Lilienthal H, Benthe C, Heinzow B, Winneke G. Impairment of schedule-controlled behavior by pre- and postnatal exposure to hexachlorobenzene in rats. Arch Toxicol 1996;70:174–81. [37] Uhnak J, Veningerova M, Madaric A, Szokolay A. Dynamics of hexachlorobenzene residues in the food chain. IARC Sci. Publ; 1986. p. 109–13. [38] Willes RF, Nestmann ER, Miller PA, Orr JC, Munro IC. Scientific principles for evaluating the potential for adverse effects from chlorinated organic chemicals in the environment. Regul Toxicol Pharmacol 1993;18:313–56. [39] Sawchenko PE, Swanson LW, Steinbusch HW, Verhofstad AA. The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat. Brain Res 1983;277:355–60. [40] Vacher CM, Fretier P, Creminon C, Calas A, Hardin-Pouzet H. Activation by serotonin and noradrenaline of vasopressin and oxytocin expression in the mouse paraventricular and supraoptic nuclei. J Neurosci 2002;22:1513–22. [41] Leibowitz SF, Eidelman D, Suh JS, Diaz S, Sladek CD. Mapping study of noradrenergic stimulation of vasopressin release. Exp Neurol 1990;110:298–305. [42] Veenema AH, Blume A, Niederle D, Buwalda B, Neumann ID. Effects of early life stress on adult male aggression and hypothalamic vasopressin and serotonin. Eur J Neurosci 2006;24:1711–20. [43] Ressler KJ, Nemeroff CB. Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders. Depress Anxiety 2000;12 (Suppl 1):2–19. [44] Jorgensen H, Kjaer A, Knigge U, Moller M, Warberg J. Serotonin stimulates hypothalamic mRNA expression and local release of neurohypophysial peptides. J Neuroendocrinol 2003;15:564–71. [45] Jorgensen HS. Studies on the neuroendocrine role of serotonin. Dan Med Bull 2007;54:266–88. [46] Jorgensen H, Knigge U, Kjaer A, Warberg J. Serotonergic involvement in stressinduced vasopressin and oxytocin secretion. Eur J Endocrinol 2002;147:815–24.

430

Z. Valkusz et al. / Physiology & Behavior 103 (2011) 421–430

[47] Knigge U, Willems E, Kjaer A, Jorgensen H, Warberg J. Histaminergic and catecholaminergic interactions in the central regulation of vasopressin and oxytocin secretion. Endocrinology 1999;140:3713–9. [48] Kimura T, Share L, Wang BC, Crofton JT. The role of central adrenoreceptors in the control of vasopressin release and blood pressure. Endocrinology 1981;108: 1829–36. [49] Kimura T, Shoji M, Iitake K, Ota K, Matsui K, Yoshinaga K. The role of central alpha 1- and alpha 2-adrenoceptors in the regulation of vasopressin release and the cardiovascular system. Endocrinology 1984;114:1426–32. [50] Leng G, Brown CH, Russell JA. Physiological pathways regulating the activity of magnocellular neurosecretory cells. Prog Neurobiol 1999;57:625–55. [51] Michaloudi HC, el Majdoubi M, Poulain DA, Papadopoulos GC, Theodosis DT. The noradrenergic innervation of identified hypothalamic magnocellular somata and its contribution to lactation-induced synaptic plasticity. J Neuroendocrinol 1997;9:17–23. [52] Bealer SL, Crowley WR. Noradrenergic control of central oxytocin release during lactation in rats. Am J Physiol 1998;274:E453–8. [53] Boersma CJ, Van Leeuwen FW. Neuron-glia interactions in the release of oxytocin and vasopressin from the rat neural lobe: the role of opioids, other neuropeptides and their receptors. Neuroscience 1994;62:1003–20. [54] Hatton GI, Perlmutter LS, Salm AK, Tweedle CD. Dynamic neuronal–glial interactions in hypothalamus and pituitary: implications for control of hormone synthesis and release. Peptides 1984;5(Suppl 1):121–38. [55] Murphy D, Levy A, Lightman S, Carter D. Vasopressin RNA in the neural lobe of the pituitary: dramatic accumulation in response to salt loading. Proc Natl Acad Sci USA 1989;86:9002–5. [56] Tweedle CD, Hatton GI. Morphological adaptability at neurosecretory axonal endings on the neurovascular contact zone of the rat neurohypophysis. Neuroscience 1987;20:241–6.

[57] Lehmann E, Hanze J, Pauschinger M, Ganten D, Lang RE. Vasopressin mRNA in the neurolobe of the rat pituitary. Neurosci Lett 1990;111:170–5. [58] Sladek CD, Kapoor JR. Neurotransmitter/neuropeptide interactions in the regulation of neurohypophyseal hormone release. Exp Neurol 2001;171:200–9. [59] Janosek J, Hilscherova K, Blaha L, Holoubek I. Environmental xenobiotics and nuclear receptors–interactions, effects and in vitro assessment. Toxicol In Vitro 2006;20:18–37. [60] Hadjab S, Maurel D, Cazals Y, Siaud P. Hexachlorobenzene, a dioxin-like compound, disrupts auditory function in rat. Hear Res 2004;191:125–34. [61] Cochon AC, San Martin de Viale LC, Billi de Catabbi SC. Phospholipid alterations elicited by hexachlorobenzene in rat brain are strain-dependent and porphyriaindependent. Comp Biochem Physiol C Toxicol Pharmacol 2001;130:199–207. [62] Egashira N, Mishima K, Iwasaki K, Oishi R, Fujiwara M. New topics in vasopressin receptors and approach to novel drugs: role of the vasopressin receptor in psychological and cognitive functions. J Pharmacol Sci 2009;109:44–9. [63] de Kloet CS, Vermetten E, Geuze E, Wiegant VM, Westenberg HG. Elevated plasma arginine vasopressin levels in veterans with posttraumatic stress disorder. J Psychiatr Res 2008;42:192–8. [64] Altemus M, Cizza G, Gold PW. Chronic fluoxetine treatment reduces hypothalamic vasopressin secretion in vitro. Brain Res 1992;593:311–3. [65] Scantamburlo G, Hansenne M, Fuchs S, Pitchot W, Marechal P, Pequeux C, et al. Plasma oxytocin levels and anxiety in patients with major depression. Psychoneuroendocrinology 2007;32:407–10. [66] Nishioka T, Anselmo-Franci JA, Li P, Callahan MF, Morris M. Stress increases oxytocin release within the hypothalamic paraventricular nucleus. Brain Res 1998;781:56–60. [67] Ryan BC, Vandenbergh JG. Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice. Horm Behav 2006;50:85–93.