gy 550 (2006) 180–185www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

Administration of antisense oligonucleotide against pro-opiomelanocortinprevents enduring hormonal alterations induced by

neonatal handling in male mice

Gabriella Galietta a,d, Alberto Loizzo a,⁎, Stefano Loizzo a, Giuseppe Trombetta a,b,Santi Spampinato c, Gabriele Campana c, Anna Capasso d, Mario Palermo e,

Irene Guarino a,f, Flavia Franconi f

a Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Roma, Italyb ASL 8 Calabria, Reggio Calabria, Italy

c Department of Pharmacology, University of Bologna, Italyd Department of Pharmacology, University of Salerno, Italy

e Unit of Endocrinology, University of Sassari, Italyf Department of Pharmacology and Excellence Centre for Biotechnology Development and Biodiversity Research, University of Sassari, Italy

Received 22 March 2006; received in revised form 18 August 2006; accepted 23 August 2006Available online 9 September 2006

Abstract

Early life events have been implicated in the programming of adult chronic diseases. Several investigations suggest that the role of earlyenvironment in influencing development mainly involves the hypothalamic–pituitary–adrenal axis. Therefore, we examined whether 1) dailyneonatal handling, applied from birth to weaning induces HPA hormones alterations in mice lasting up to the adult age; and 2) if the administrationof an antisense oligodeoxynucleotide versus pro-opiomelanocortin (As-POMC) prevents hormonal alterations observed in previously handledmice (Handled). In the adult phase (90 days), Handled are overweight and have higher basal plasma immuno-reactive (ir)-corticosterone andadrenocorticotropin (ir-ACTH), and higher pituitary ir-ACTH; while they have lower hypothalamic ir-ACTH and corticotropin-releasinghormone (ir-CRH) in comparison with the non-handled mice. As-POMC (0.05–0.1 nmol/g body weight per day) administered during the sameperiod dose-dependently prevents the increase in body weight, in plasma ir-corticosterone, ir-ACTH, and pituitary ir-ACTH, also preventing thedecrease in hypothalamic ir-CRH and ir-ACTH; while the mismatch oligonucleotide is nearly inactive. This data indicates that pharmacologicaltreatment in neonatal life may have enduring effects, reducing the alterations in hormonal homeostatic programming mechanisms induced by earlyrepeated handling.© 2006 Elsevier B.V. All rights reserved.

Keywords: Hypothalamus–pituitary–adrenal axis; Corticotropin-releasing hormone; Adrenocorticotropin; Corticosteroid; Neonatal programming; Pro-opiomela-nocortin antisense

1. Introduction

Development continues after birth, and therefore early lifeevents can influence the maturation of the nervous system. Thephenomenon dubbed “programming” has received much atten-

⁎ Corresponding author. Istituto Superiore di Sanità, viale Regina Elena, 299;00161, Roma, Italy. Tel.: +39 06 4990 2882; fax: +39 06 4938 7100.

E-mail address: alberto.loizzo@iss.it (A. Loizzo).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.08.072

tion because it could be the root of some adult diseases (Godfrayand Baker, 2001; O'Regan et al., 2001; Singhal and Lucas,2004). Central nervous system regions governing vegetativefunctions are subject to programming, among these the mostthoroughly studied is the hypothalamic–pituitary–adrenal (HPA)axis (Ng, 2000; O'Regan et al., 2001; Matthews, 2002; Williamset al., 2003; Seckl, 2004). Classically, HPA axis involves hypo-thalamic parvocellular paraventricular nucleus, which releasescorticotropin-releasing hormone (CRH) that stimulates the an-terior pituitary to induce release of adrenocorticotropin (ACTH);

181G. Galietta et al. / European Journal of Pharmacology 550 (2006) 180–185

in turn the pituitary hormone induces glucocorticoid secretionfrom the adrenal cortex (O'Regan et al., 2001). Glucocorticoidsregulate theHPA axis by a feedbackmechanism at the level of thepituitary, the hypothalamic, and the supra-hypothalamic limbicstructures, particularly the hippocampus and amygdala (Brad-bury et al., 1993, Rhen and Cidlowski, 2005). Persistent changesin feedback mechanisms of HPA axis may occur (Matthews,2002) for example through the decreased expression of brainglucocorticoid (mineralcorticoid) receptors (Edwards and McIn-tyre Burnham, 2001; De Kloet, 2003; Ladd et al., 2004). Inte-restingly, neonatal glucocorticoids treatments negatively affectprogramming, thus influencing cognitive processes in childrenand increasing the risk of developing hypertension in adolescents(Doyle et al., 2000).

The ‘pharmacology’ of programming has usually been stud-ied examining the short- and long-term consequences of theexposure of newborns to exogenous agents (Jones et al., 1999;Williams et al., 2000; Feng et al., 2001; Sanabria et al., 2002;Garofolo et al., 2003; Nicosia et al., 2003; Williams et al.,2003; Zhang et al., 2003; Yilmazer-Hanke et al., 2004; Fran-coni et al., 2004). Here we propose the use of antisenseoligonucleotides, which are designed to bind to selected targetmRNA molecules by Watson–Crick base pairing, which re-sults in the inhibition of mRNA processing or translation; theyhave already been proposed to treat cancer and neurologicaldiseases (Jansen and Zangemeister-Wittke, 2002; Forte et al.,2005). However, their effective intracellular delivery remainsan important issue for their clinical application, but antisensephosphorothioates cross the blood–brain barrier (Banks et al.,2001). Additionally, we have previously shown that parenteraladministration of the phosphorothioate antisense mRNAversus ACTH in neonates significantly reduces the anteriorpituitary ir-ACTH, as well as plasma corticosterone in new-born mice, whereas its mismatch analogue (MS-ACTH) isinactive (Loizzo et al., 2003).

Very recently, it has been shown that repeated neonatalhandling (short-lasting maternal deprivation plus sham injec-tion) induces overweight and elevated plasma ir-ACTH andcorticosterone in adult male mice (Loizzo et al., 2006). There-fore, it has been investigated whether neonatal administrationof antisense oligodeoxynucleotide versus pro-opiomelanocor-tin (As-POMC) is able to prevent enduring effects induced byhandling.

2. Methods

2.1. Animals

Experiments were carried out in accordance with theguidelines of the Council of European Communities 86/609/EEC and the protocols were approved by the BioethicalCommittee of the Istituto Superiore di Sanità (Roma, Italy).Animals were housed in light (12 h on and 12 h off), tem-perature (20±2 °C) and humidity (55±5%) controlled envi-ronments, with food (Mucedola S.r.l., Settimo Milanese, Italy)and water available ad libitum. Animals were handled aspreviously described in Loizzo et al. (2003), Franconi et al.

(2004) and Loizzo et al. (2006). Briefly, different series (totalnumber 18) of multiparous pregnant mice from the CD-1outbred strain (Charles River, Calco-Lecco, Italy) were re-ceived, and about 12 h after birth, male pups of similar weightwere selected and six pups per litter were randomly puttogether and cross-fostered, and randomly assigned to one ofthe groups: 1) non-neonatal-handled mice (Controls): the pupswere left undisturbed with their mothers, except for cagecleaning twice a week; 2) vehicle-treated handled mice (Han-dled): for 21 days, the pups were removed daily (10 min) fromthe home cage and grouped in a container with fresh beddingmaterial and injected s.c. with sterile saline (1 ml/kg bodyweight); 3) As-POMC-treated handled mice pups were han-dled as in the vehicle groups but they were treated with As-POMC (0.05 and 0.1 nmol/g body weight; As-Treated 0.05Handled and As-Treated 0.1 Handled, respectively); 4) Mm-POMC-treated handled mice (Mm-Treated Handled): thesepups were handled as in the vehicle groups and received Mm-POMC (0.1 nmol/g body weight).

After weaning, the animals were re-housed three per cage,and left undisturbed until being sacrificed, with the exception ofbody weighing and cage cleaning.

2.2. Drugs

As-POMC and Mm-POMC were produced by EUROBIOLaboratories (Les Ulis Cedex, France) under the direction ofone of us (S.S.). Briefly, the 21-base sequence of As-POMC andMm-POMC were respectively:

5′-TCT GGC TCT TCT CGG AGG TCA-3′, and 5′-TGTGCC TCT TTC CGG TGG ACA-3′

It has been previously reported that these antisense andanalogous products reduced synthesis of some POMC-derivedhormones both in vitro or in vivo. Molecular differences in-duced by amino acid transposition in Mm-POMC (in boldcharacters) were effective in modulating specific hormonaleffects (Spampinato et al., 1994; Loizzo et al., 2003).

In order to avoid the potential confounding factor of a hugedecrease in the activity of HPA axis we selected doses thatwere able to reduce the activity increase due to handling, butleft the basal level of POMC-derived molecules unaltered(Fig. 1, A and B). To evaluate the effects induced by short-lasting As-POMC treatment, in the pilot experiment the ani-mals were sacrificed after the 21st day of treatment in order tomeasure plasma POMC-derived peptides. It is evident that at21 days of life, plasma ir-ACTH and ir-β-endorphin levelswere consistently higher in Handled than in As-Treated 0.05Handled and in As-Treated 0.1 Handled mice (Fig. 1, A andB). Interestingly, plasma levels of ACTH and β-endorphin inMm-Treated Handled were practically identical to those mea-sured in Handled, thus suggesting the specificity of antisensetreatments.

2.3. Plasma and tissue preparations

Non-fasting animals (to avoid possible stress induced byfasting) were rapidly sacrificed between 09:00 and 12:00 h

Fig. 2. Body weight (g) at different ages (days) in the experimental groups.Abscissa: age in days. Ordinate: values are weight means in g of 12 animals pergroup±S.E.M.; C: Controls, H: Handled, Mm: Mismatch-Treated and Handled,As 0.05: Antisense-Treated (0.05) and Handled, As 0.1: Antisense-Treated (0.1)and Handled; +: Pb0.001 versus H; ×: Pb0.001 versus Mm; ■: Pb0.05 ver-sus Mm.

Fig. 1. Plasma levels of ir-β-endorphin (ng/ml) (A) and ir-ACTH (pg/ml) (B)measured in 21-day-old male mice. C: Controls, H: Handled, Mm: Mismatch-Treated and Handled, As 0.05: Antisense-Treated (0.05) and Handled, As 0.1:Antisense-Treated (0.1) and Handled. A) Values are mean±S.E.M. of at least 5animals; +: Pb0.01 versus C; ×: Pb0.01 versus H; ■: Pb0.05 versus Mm.B) Values are mean±S.E.M. of at least 5 animals; +: Pb0.001 versus C; ×:Pb0.001 versus H; ■: Pb0.0001 versus Mm, ●: Pb0.001 versus As 0.05, ▴:Pb0.001 versus As 0.1.

Fig. 3. Plasma levels of ir-ACTH (pg/ml) (A), and ir-corticosterone (ng/ml) (B)measured in 90-day-old male mice. Values are mean±S.E.M. of at least 5animals; C: Controls, H: Handled, Mm: Mismatch-Treated and Handled, As0.05: Antisense-Treated (0.05) and Handled, As 0.1: Antisense-Treated (0.1)and Handled; +: Pb0.0001 versus C; ×: Pb0.001 versus H; ■: Pb0.0001versus Mm, ●: Pb0.001 versus As 0.05.

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noon. Trunk blood was collected in either ice-chilled heparin-ized or in EDTA-containing tubes according to each hormone-specific assay protocol and spun at 3500 ×g for 10 min at 4 °C.Plasma was stored at −80 °C until assayed. The pituitary andhypothalamus were dissected on dry ice and immediately storedat −80 °C. The day of assay, according to Spampinato andGoldstein (1983), tissues were extracted in 0.1 mM acetic acid90 °C and used throughout.

2.4. Immune-reactive (ir-)ACTH, ir-CRH, ir-corticosterone andir-β-endorphin determinations

Tissue and plasma ir-CRH and ir-ACTH were measuredwith a double-antibody precipitation radioimmunoassay (Nich-olson et al., 1984). The human antibodies (Dr. A.F. Parlow,Harbor-UCLAMedical Center, USA) recognised mouse ACTH(1–39) and mouse CRH (Bachem AG, Bubendorf, Switzerland)and did not cross-react with other peptides derived from thePOMC and CRH precursors. The detection limit for ACTH was6±0.23 pg/ml; IC50 was 229±26 pg/ml (mean±S.D.); whileCRH detection limit was 3±0.12 pg/ml; IC50 was 14.7±1.5(mean±S.D.). [125I] iodotyrosyl2-ACTH (1–39) [125I] iodo-tyrosyl0-CRH were purchased from Amersham Biosciences(Milano, Italy), and Bachem AG (Bubendorf, Switzerland).

Ir-corticosterone and ir-β-endorphin-like material wereassayed with RIA kits (ICN, Costa Mesa, USA; Peninsula

Laboratories Inc, Belmont, CA, USA, respectively) as pre-viously described in Fontana et al. (1997). The detection limitfor ir-β-endorphin-like material was 10 pg/ml and IC50 was50±2 pg/ml (mean±S.D.).

All assays were measured in duplicate and all determina-tions had intra-assay and inter-assay variations less than 2%.Tissue proteins were measured according to Bradford (1976).

Fig. 5. Hypothalamic levels of ir-CRH (pg/μg protein) measured in 90-day-oldmale mice. Values are mean±S.E.M. of at least 5 animals; C: Controls, H:Handled, Mm: Mismatch-Treated and Handled, As 0.05: Antisense-Treated(0.05) and Handled, As 0.1: Antisense-Treated (0.1) and Handled; +: Pb0.0001versus C; ×: Pb0.0001 versus H, ■: Pb0.0001 versus Mm, ●: Pb0.001 ver-sus As 0.05.

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2.5. Statistics

Values are mean±S.E.M. Statistical differences were calcu-lated with an analysis of variance followed by Newman–Keulsmultiple comparisons and P≤0.05 was considered significant.

3. Results

3.1. General observations

No animal deaths were observed during the experimentaltime. None of the neonatal handling and treatments modified thegrowth increment curves during the suckling period (Fig. 2). At60 and 90 days of age, the body weight in Handled was con-sistently higher than in Controls. The increase in body weightinduced by neonatal handling was respectively decreased andabolished in As-Treated 0.05 Handled and in As-Treated 0.1Handled, while the weight of Mm-Treated Handled mice did notdiffer from Handled (Fig. 2). Mm-Treated Handled and As-Treated Both Doses Handled did not exhibit any overt physicalor behavioural abnormalities.

3.2. Plasma levels of ir-ACTH and ir-corticosterone in 90-day-old mice

At 90 days of age, plasma levels of ir-ACTH and ir-corti-costerone were higher in Handled than in Controls. The increase

Fig. 4. Pituitary (A) and hypothalamic (B) levels of ir-ACTH (pg/μg protein)measured in 90-day-old male mice. Values are mean±S.E.M. of at least 5animals; C: Controls, H: Handled, Mm: Mismatch-Treated and Handled, As0.05: Antisense-Treated (0.05) and Handled, As 0.1: Antisense-Treated (0.1)and Handled; +: Pb0.0001 versus C; ×: Pb0.0001 versus H, ■: Pb0.0001versus Mm, ●: Pb0.001 versus As 0.05.

was dose-dependently prevented by neonatal As-POMC but notby Mm-POMC administration (Fig. 3, A and B).

3.3. Effect induced by As-POMC treatment on hypothalamicand pituitary ir-ACTH levels in 90-day-old mice

The pituitary ir-ACTH level was significantly higher inHandled and Mm-Treated Handled than in Controls. Neonataltreatment with As-POMC dose-dependently lowered the pitui-tary ir-ACTH levels in adult animals (Fig. 4A).

Conversely, adult Handled and Mm-Treated Handled ani-mals had hypothalamic ir-ACTH levels consistently lower thanControls. Again, neonatal As-POMC treatment prevented thedecrease induced by neonatal handling in a dose-related fashion(Fig. 4B). This was in agreement with the hypothesis that theneonatal treatment with As-POMC could prevent dysregulationof the HPA axis feedback mechanisms in adult animals.

3.4. Effect induced by As-POMC treatment on hypothalamic ir-CRH levels in 90-day-old mice

Adult animals, neonatally handled and treated with vehicleor with Mm-POMC, had hypothalamic ir-CRH consistentlylower than non-handled animals. Again, the decrease in ir-CRHlevel was dose-dependently prevented by As-POMC (Fig. 5).

4. Discussion

Neonatal handling significantly increases body weight, plas-ma ir-corticosterone and ir-ACTH in adult mice, as previouslydescribed in Loizzo et al. (2006). Here, it has also been shownthat Handled adult mice have a higher pituitary ir-ACTH and alower hypothalamic ir-ACTH and ir-CRH levels than Controls.More interestingly, all these alterations are dose-dependentlyprevented by neonatal As-POMC administration. Resultsgathered from our Handled indicate an overactivity and altered

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feedback regulation of HPA axis, that remind one of theglucocorticoid resistance phenomena. Glucocorticoid resistanceis sustained by different mechanisms, such as a variation of theexpression in glucocorticoid receptors and an activation ofMAP-kinases, which phosphorylate the glucocorticoid receptorand thereby inhibit the glucocorticoid signalling (Rhen andCidlowski, 2005).

HPA axis can be permanently programmed during the deve-lopment (Levine, 1957). Indeed, present findings in our Han-dled adult mice (low level of hypothalamic ir-CRH, highplasma levels of ir-ACTH and ir-corticosterone ) suggest thatthe feedback mechanism at the hypothalamic level is function-ally active, while not working at the pituitary level, because thepituitary ir-ACTH is still high in the presence of high plasmacorticosterone. In addition, plasma levels of ir-ACTH at 21 and90 days of age are very similar, suggesting that this (pre-sumably) irreversible activation starts at a very early age, i.e.,probably before weaning.

More interestingly, the pituitary (ir-ACTH) and adrenal (ir-corticosterone) activation are dose-dependently inhibited byneonatal As-POMC administration, as a consequence of a spe-cific action on POMC mRNA. The real role of As-POMCtreatment in preventing neuroendocrine activation is furthersupported by results obtained with Mm-POMC, which differsfrom the As-POMC in six positions and is practically inactive,suggesting – according to Myers et al. (2003) and Kurreck(2003) – a specific antisense-induced effect.

Therefore, neonatal As-POMC administered parenterallyduring a period when the blood–brain barrier is not fully de-veloped (Banks et al., 2001; Clot-Faybesse et al., 2000), mayalso reach brain areas and “reset” HPA axis not only at thepituitary level, but apparently also at the hypothalamic level.This is also supported by the fact that both groups treated withAs-POMC are not overweight, thus suggesting a link betweenbeing overweight and HPA axis alterations, as happens inpatients with Cushing syndrome (Salehi et al., 2005). In mice,much neuroendocrine development occurs in the post-natalperiod (Sapolsky and Meaney, 1986) which could be importantin explaining the efficacy of neonatal antisense treatments.

At this time, we do not know whether ir-β-endorphin ismodified in adults, but in previous experiments performed withthe same experimental paradigm neonatal treatment of nalox-one, an antagonist of opioid receptors, prevented overweightand immunological alterations induced by neonatal handling(d'Amore et al., 1996; Loizzo et al., 2002). Nevertheless,neonatal naloxone (1 mg/kg per day) is not as effective as As-POMC in preventing variations in HPA axis (Loizzo et al.,unpublished observations) in neonatally handled adult mice.

All this data suggest that neonatal As-POMC treatmentreduces hormonal alterations induced by neonatal handlingthrough a specific action on POMC mRNA. Generally, anti-sense drugs are not considered very safe (Pirollo et al., 2003). Inour experimental conditions no apparent toxicity was detected.Nevertheless future research should specifically focus on thesafety of AS-POMC treatment.

HPA axis alterations are involved in the pathogenesis ofseveral adulthood diseases, such as generalised psychiatric

diseases (anxiety disorders, major depression) (Huttunen andNiskanen, 1978; Meijer, 1985; Bandelow et al., 2005), obesity,metabolic syndrome X, type 2 diabetes mellitus and cardiovas-cular diseases (Clark, 1998; Godfray and Baker, 2001). Al-though caution must be used in extrapolation among species, itseems important to remember that similar HPA axis overactivityoccurs in low birth weight humans, and may be, at least in part,the explanation behind the “low birth weight” phenotype (Phil-lips et al., 2000), which is associated with an increased preva-lence in cardiovascular diseases, insulin resistance, and obesityin the adult life of humans (Clark, 1998; Phillips et al., 1998;Levitt et al., 2000; Phillips et al., 2000; Godfray and Baker,2001; Reynolds et al., 2001; Singhal and Lucas, 2004). Lowbirth weight and stressful events in childhood are also known tobe major risk factors contributing to the development of neu-rodevelopmental disorders and depressive illness, respectively(Barker et al., 1995; Burd et al., 1999; Heim and Nemeroff,2001). It appears that alleviating HPA axis imbalance in theneonate may decrease risk factors for numerous diseases; inconclusion, As-POMC or other treatments with similar effectson the HPA axis may be a novel approach for counteracting thepreviously described risks.

Acknowledgements

This work was supported by NIH-ISS Research Project“Gender differences in seizure sensitivity: the role of steroidsand neurosteroids”; with the contribution of Fondazione EnteCassa di Risparmio di Firenze, Ente Fondazione Cassa di Ris-parmio di Pistoia e Pescia and GIO.I.A. Foundation onlus. Weexpress deep gratitude to Dr Maurizio Mian for his advice fordiscussion, to Mrs Erin Griffin for the English revision of themanuscript, and to Mrs Carla Campanella for her editorial work.Mr Stefano Fidanza and Mr Adriano Urcioli provided valuableanimal care.

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