Neurotoxicology and Teratology 46 (2014) 32–39

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Neurotoxicology and Teratology

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Sex-dimorphic effects of gestational exposure to the organophosphateinsecticide chlorpyrifos on social investigation in mice

Alessia De Felice a,b, Aldina Venerosi a, Laura Ricceri a, Mara Sabbioni a,c, Maria Luisa Scattoni a,Flavia Chiarotti a, Gemma Calamandrei a,⁎a Section of Neurotoxicology and Neuroendocrinology, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italyb Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00161 Rome, Italyc Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00161 Rome, Italy

⁎ Corresponding author at: Department of Cell BioloSuperiore di Sanità, Viale Regina Elena 299, I-00161 Romfax: +39 064947821.

E-mail addresses: alessiadefelice5@gmail.com (A. De F(A. Venerosi), laura.ricceri@iss.it (L. Ricceri), mara.sabbionmarialuisa.scattoni@iss.it (M.L. Scattoni), flavia.chiarotti@gemma.calamandrei@iss.it (G. Calamandrei).

http://dx.doi.org/10.1016/j.ntt.2014.09.0020892-0362/© 2014 Published by Elsevier Inc.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 March 2014Received in revised form 8 September 2014Accepted 10 September 2014Available online 28 September 2014

Keywords:ChlorpyrifosSocial investigationSocial discriminationOlfactory discriminationSex differencesRodents

Several pieces of evidence from animal and human studies indicate that the organophosphate insecticide chlor-pyrifos (CPF) acts as a developmental neurotoxicant at environmentally relevant doses, and it is possiblyendowedwith endocrine-disrupting activity. Data collected in rodentmodels show that developmental exposureto CPF at sub-toxic doses induces long-lasting and sex-dimorphic changes in social and investigative responses inexposed offspring. The aim of this study was to evaluate the effects of gestational CPF treatment on social and ol-factory discrimination in adult mice of both sexes. Pregnant CD1 out-bred mice were exposed to CPF per os ongestational days (GD) 14–17 at the sub-toxic dose of 6 mg/kg/bw. At adulthood, male and female offspringunderwent the same experimental paradigms, namely i) a social discrimination test wheremice were presentedwith a simultaneous binary choice between a novel conspecific and a familiar one, and ii) an olfactory habitua-tion/dishabituation test to evaluate their capability to discriminate between odors with different eco-ethological salience (non-social vs. social odors).Results showed that in the social discrimination test prenatal CPF primarily affected the female sex by raising theinvestigation time in females to the same levels as found in vehicle- and CPF-exposed males. The ability to dis-criminate between a familiar and a novel social mate was not affected by CPF in either sex. In the olfactory habit-uation/dishabituation test, mice of both sexes successfully discriminated non-social from social odors regardlessof the prenatal treatment received.These results confirm previous evidence indicating that developmental exposure to CPF causes long-lasting andsex-dimorphic changes in responsiveness to social cues, in the absence of significant impairment of social and ol-factory discrimination capacity. These findings are discussedwithin the framework of recent data pointing to thelimbic/hypothalamic circuitry and steroid hormonal regulations as possible targets for CPF neurotoxicity.

© 2014 Published by Elsevier Inc.

1. Introduction

Chlorpyrifos (CPF) is the most used non-persistent organophos-phate (OP) pesticide worldwide in both agricultural and urban commu-nities. While the acute neurotoxicity of this chemical is associated withsystemic and brain acetylcholinesterase (AChE) inhibition, an increas-ing body of experimental data suggests that, at low doses, CPF also tar-gets non-cholinergic mechanisms (Eaton et al., 2008; Slotkin andSeidler, 2012).

gy and Neurosciences, Istitutoe, Italy. Tel.: +39 0649902106;

elice), aldina.venerosi@iss.iti@hotmail.it (M. Sabbioni),iss.it (F. Chiarotti),

Several epidemiological studies to date support the hypothesis thatat low, environmentally relevant doses, CPF acts as a developmentalneurotoxicant. Analysis of longitudinal birth cohorts from either agri-cultural or urban communities found associations between prenatal ex-posure to CPF asmeasured in cord blood and a higher risk ofmental andmotor delay, pervasive developmental disorder (PDD), and hyperactivebehaviors in exposed children (Rauh et al., 2006).

The mechanisms by which CPF affect neurobehavioral developmentin exposed children are unclear, as are the possible delayed effects ofthis chemical at older ages. Studies in rodents demonstrated that atdoses devoid of systemic toxicity, developmental exposure to CPF inter-feres with DNA synthesis, neuronal differentiation, synaptogenesis, andaffects the expression levels of critical genes involved in brain develop-ment (Betancourt et al., 2006; Crumpton et al., 2000; Dam et al., 1998).Furthermore, fetal exposure to subtoxic doses of CPF alters neural sys-tems beyond cholinergic transmission, such as serotonergic and dopa-minergic neurotransmission, in a sex-dimorphic fashion (Slotkin and

33A. De Felice et al. / Neurotoxicology and Teratology 46 (2014) 32–39

Seidler, 2007). Numerous rat studies have shown long-term effects onbehavior after developmental exposure to subtoxic doses of CPF(Aldridge et al., 2004; Aldridge et al., 2005; Dam et al., 1999; Raineset al., 2001; Slotkin et al., 2002). Specifically, prenatal CPF exposurecauses multiple behavioral alterations in rats tested from adolescenceto adulthood; locomotor activity habituation andworking and referencememory are affected with significant damping of sex differences in CPFexposed females (Levin et al., 2002). Similarly in themouse species, CPFdoses below the threshold for adverse effects on fetal growth or viabilityandmaternal toxicity induce sex-dimorphic and long-term changes in anumber of behavioral end points. In particular, in addition to increasingmotor activity in themales, CPF targets different items of the social rep-ertoire of laboratorymice in both sexes, including social recognition andpup-directed responses in virgin females (Ricceri et al., 2006), aggres-sive interactions between males to achieve social rank (Ricceri et al.,2006), social recognition (Venerosi et al., 2006), and nest defense re-sponse in lactating females (Venerosi et al., 2009). We have alsoshown that CPF has long lasting effects on the neurohormones implicat-ed in the modulation of social and affective responses such as oxytocin(OT) and vasopressin (AVP) (Tait et al., 2009). Notably, hypothalamicneuropeptides are considered components of a sex-dimorphic hypotha-lamic–limbicmicronetmodulating social responses in rodents (Choleriset al., 2003), and regulating social recognition at the level of the olfacto-ry system in rodents (Bielsky and Young, 2004; Wacker and Ludwig,2012).

On the basis of this experimental evidence, we hypothesized thatone of the possible mechanisms underlying CPF behavioral toxicitymay be the interference with endocrine factors and/or sexually dimor-phic features of brainmaturation (Venerosi et al., 2012). As social inves-tigation and discrimination are markedly sex-dimorphic in rodents, weexpect to find sex-dependent vulnerability to CPF effects by assessingadult mice of both sexes with the same experimental paradigm. Specif-ically in the present study, we evaluated the effects of gestational CPFexposure on social investigation and discrimination by applying a socialdiscrimination testwhere two social stimuli are repeatedly presented tothe same animal before the final simultaneous binary choice between anovel conspecific and a familiar one (Choleris et al., 2003). Additionally,in order to verify the possibility that CPF alters social responses by inter-fering with the detection and/or processing of olfactory cues, weassessed the same mice in a habituation/dishabituation olfactory testwhere non-social (water, vanilla, almond) or social (same-sex urine,opposite-sex urine) odors were presented sequentially to mice to eval-uate their ability to distinguish between odorswith different ethologicalvalue and to habituate to them (Yang and Crawley, 2009). CPF or its ve-hiclewere administered at the sub-toxic dose of 6 mg/kg/bwby oral ga-vage from gestational day (GD) 14 to 17 to pregnant mice of the CD1strain.

2. Materials and methods

2.1. Subjects and treatment

Forty male and female mice of the out bred Swiss-derived strain(CD1, Harlan, S. Pietro al Natisone, Italy), were housed in pairs in breed-ing cages (polycarbonate cages 33× 13× 14 cm)with a 12-h light–darkcycle (lights on at 8 pm) with free access to food (enrichment standarddiet for mice, from Mucedola, Settimo Milanese, Italy) and water.

Females were inspected daily for the presence of the vaginal plug(gestational day 0). The stud was removed 10 days after the discoveryof the vaginal plug. On GD14, 26 pregnant females were randomlyassigned to one of the two prenatal treatments [vehicle (Veh), CPF].CPF (Chem. Service, West Chester, PA) was dissolved in peanut oil(Veh) to provide rapid and complete absorption. CPF (in a volume of0.1 ml/10 g at a dose of 6 mg/kg/bw) or its vehicle was administeredto pregnant females from GD 14 to 17 by intraoral gavages. Extensivework by Slotkin's group in rats has shown a greater sensitivity of late

gestational phases (for doses comprised between 1 and 5 mg/kg/bw)for the effects of prenatal CPF exposure on both neural systems and be-havior (Aldridge et al., 2005; Qiao et al., 2003).

This same late gestation exposure window and the dose used in ourpresent studywas found to produce significant effects on adult social re-sponses and hypothalamic neuropeptide levels in mice (Ricceri et al.,2006; Tait et al., 2009). Notably, the lowest observed adverse effectlevel (LOAEL) established by the EU for neurotoxicity in rats after chron-ic exposure is 10 mg/kg/bw/day, although most of the developmentalneurotoxicity studies in rats and mice found delayed behavioral effectsat doses comprised between 1 and 6 mg/kg/bw/day. Estimated CPF ex-posure in the general population including children and women ofchildbearing age today is primarily through diet and in the 10−1 to10−3 μg/kg-d dose range (Li et al., 2012), although much higher expo-sure might occur in the fetus in areas with intensive pesticide use(Ostrea et al., 2002).

The 6 mg/kg dose is safe with respect to the reproductive perfor-mance of treated dams (pregnancy length, number of pups at delivery,sex ratio), and it does not induce overt toxic symptoms in dams ormajor effects on pups' health parameters such as weight at deliveryand impairment of growth rate (Ricceri et al., 2006). Following CPFdose administration to pregnant mice after applying the same treat-ment schedule of the present study, we found no effects on brainAChE activity and a mild transient inhibition (20% of control values) inserum AChE activity in offspring when measured at birth 24 h afterthe last exposure (Ricceri et al., 2006). We cannot exclude that limitedbut significant brain AChE inhibition could have been present at earliertime points and recovery could have occurred by 24 h following expo-sure as previously indicated in the rat species by (Mattsson et al.,2000). Of the twenty-six pregnant females treated, four females (twoVeh and two CPF treated) gave birth on GD 17 and were thus excludedfrom further study. A total of twenty-two litters (12 Vehicle-treated and10 CPF-treated) were used. On the day of birth, number of pups deliv-ered, overall weight of the litter, and sex ratio were recorded to verifythe absence of CPF effects on reproductive performance by consideringthe body weight of each pregnant female before the beginning of treat-ment as a covariate. We assessed the sex of the pups by evaluation ofano-genital distance and litters were culled to a maximum of 10 pupswhile always trying to maintain a comparable number of pups of thetwo sexes within the litter (0.6 ≤ sex ratio ≤ 1.5). Offspring wereweaned on postnatal day 23, housed in cages containing littermates ofthe same sex, and left undisturbed until the beginning of the behavioralassessment. At adulthood, one female and onemale from each Veh (12)and CPF-treated (10) litter underwent the social discrimination test,while one–two females and one–two males from the same litterswere assessed in the olfactory habituation/dishabituation test.

All experiments on animals were performed according to theEuropean Community Council Directive 2010/63/EU and to Italian Leg-islation on Animal Experimentation (Legislative Decree 116/92).

2.2. Social discrimination paradigm

On postnatal day 70, both females (Veh, n=12; CPF, n=9; one fe-male was excluded due to loss of video recorded data) and males (Veh,n= 12; CPF, n= 10) from each treatment underwent a social discrim-ination test as described by Choleris et al. (2006). The test was per-formed during the dark phase of the light/dark cycle in a novelpolycarbonate cage (48 × 27 × 21 cm) where the experimental subjectunderwent a five trial social discrimination test between two socialstimuli (age- and sex-matched CD1 mice).

In each trial (T1–T5), twowired cylinders (Galaxy Cup, Kitchen Plus,http://www.kitchen-plus.com; diameter 10 cm, height 10.5 cm), eachcontaining a stimulus mouse from different cages, were placed in twoopposite sides of the test cage. The use of wired cylinders allowed forpassage of olfactory cues while preventing direct interactions betweenstimulus and experimental mice. This ensured that experimental mice

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did not deposit their own scent onto stimulus mice, while still elicitinghigh social interest and investigation from experimental mice.

Each trial lasted 5 min. In trials T1–T4, the same two stimulus micewere placed in identical locationswithin the experimental cage, where-as in trial T5 oneof the two stimulusmicewas novelwhile the otherwasthe same familiar mouse placed in the same location as in trials T1–T4.The location of the novel mouse at T5 was randomized across subjects.The T1–T5 trials were separated by 15-min intervals during which theexperimental subjects remained in the test cage in the presence of thetwo empty cylinders used during T1–T5 trials. Importantly, in all teststhe location of stimulus mice was kept constant to make spatial cues ir-relevant to the task. Two procedures were adopted to limit bias due tonovelty and stress effects: i) before testing, the experimental subjectswere left undisturbed for one hour in the experimental cage with twoempty wired cylinders to habituate to them; ii) stimulus mice were ha-bituated to cylinders several days prior to the day of the test until theyspontaneously entered them with no observable distress.

A detailed analysis of the mouse behavior based on the species'ethogram was performed to assess both specific social and non-socialresponses to the stimulus mice. To this aim, trials (T1–T5) were videorecorded and observations were analyzed using the Observer VideoAnalysis software (Noldus Information Technology, Wageningen, theNetherlands) by a trained experimenter blind to the treatment receivedby each mouse. The time spent in social investigation (sniffing everybody part of the stimulus mice through thewired cylinders) was scoredseparately on each of thefive trials. In T1–T4where the familiarity of themice at each trial was equivalent, animals were expected to investigateeach stimulus mouse around 50% of the total time (random choice)showing a similar habituation profile to both stimulus mice; while inT5, if the mice can successfully discriminate between the novel andthe familiar stimulus mouse, the proportion of time spent investigatingthe novel stimulusmouse should be significantly higher than that spentin investigating the familiar mouse. Thus, as test conditions changedmarkedly between T1–T4 and T5 sessions, datawere analyzed separate-ly for T1–T4 (social habituation) and T5 (social discrimination).

Immobility, self-grooming, wall-rearing, and digging behaviors(both frequency and duration) were also recorded throughout the five5 min-trials.

2.3. Olfactory habituation/dishabituation

The olfactory habituation/dishabituation test analyzes the animal'stendency to investigate novel smells and the mouse's ability to detectand differentiate odorswith different eco-ethological values. The exper-imental paradigm applied here is a slightly modified version of the pro-cedure described by Yang and Crawley (2009).

A total of thirty-two females (Veh, n=16; CPF, n=16) and twenty-sixmales (Veh,n=14; CPF, n=12)were used for this test. The testwasperformedduring the dark phase of the light/dark cycle in a polycarbon-ate cage (33 × 13 × 14 cm) identical to the home cage but with cleansawdust, whereas the experimental subject was exposed to a cottonswab laterally introduced through the cage lid. Each mouse was intro-duced to the experimental cage for 1 h prior to testing to habituatethem to the setting. Females were tested when not in estrous asassessed by visual observation of the vagina (Byers et al., 2012). Thetest was characterized by sequential exposure to different non-socialand social odors. Each trial lasted 2 min, and each odor was presentedin a consecutive 3-trial (T1, T2, and T3) sequence. Sequences of threeidentical swabs determine habituation to the same odor, whileswitching to another odor determine dishabituation. Habituation wasdefined as the decrease from trial 1 to trial 3 in time spent sniffing thecotton swab soaked with a given non-social or social odor.Dishabituation was defined as the increase in time spent sniffing thecotton swab soaked with a novel odor, following three successive pre-sentations of the previous social or non-social odor.

Odor presentationwas performed by inserting into the cage a cottonswab soaked with one of the following non-social odors: a) water;b) vanilla extract (1:100 dilution); c) almond extract (1:100 dilution),or cotton swab absorbed with a social odor obtained wiping the cottonswab in a zigzag pattern across the bottom surface of a cage of unfamil-iar mice of d) same strain, same sex; e) same strain, different sex. Socialodors were collected from one-week-old bedding of either group-housed females or individually-housed males. The order of swab pre-sentationwas:water 1,water 2,water 3, vanilla 1, vanilla 2, vanilla 3, al-mond 1, almond 2, almond 3, social odor same sex 1, social odor samesex 2, social odor same sex 3, social odor different sex 1, social odor dif-ferent sex 2, social odor different sex 3. Time spent sniffing each odorwas scored when the animal oriented toward the cotton tip with itsnose being within 2 cm of the cotton swab.

2.4. Statistical analysis

Reproductive performance datawere analyzed by analysis of covari-ance (ANCOVA) with treatment as the between-subject fixed factor (2levels), and body weight of pregnant females before the beginning oftreatment as the covariate. Behavioral data from the social discrimina-tion test during trials (T1–T4) were analyzed by analysis of variance(ANOVA) with prenatal treatment (2 levels) as the between-litterfixed factor, sex (2 levels) as the within-litter fixed factor, and trials asthe repeated measures factor (4 levels). Social investigation in T5 wasanalyzed separately applying the ANOVA model with prenatal treat-ment (2 levels) as the between-litter fixed factor, sex (2 levels) as thewithin-litter fixed factor, and familiarity/novelty (time spent in investi-gating either the familiar or the novel stimulus mouse; 2 levels) as thewithin-subject fixed factor.

In the olfactory habituation/dishabituation paradigm, more than 1female and 1 male were used per litter. Additional animals were select-ed randomly and the litter effect was taken into account for calculatingthe mean square of errors in the ANOVA. Time sniffing the cotton swabsoaked with different non-social and social odors was analyzed by theANOVA model with prenatal treatment (2 levels) as the between-litter fixed factor, sex (2 levels) as the within-litter fixed factor, andodors (5 levels) and trials (3 levels) as the repeated measures factors.Greenhouse–Geisser correction (G–G) was used to deal with violationof the sphericity assumption when testing repeated measures factors.Multiple comparisons were performed using Tukey's test that can alsobe used in the absence of a significant (p b 0.05) interaction tominimizeboth Type I and Type II errors.

3. Results

3.1. Reproductive performances

In agreement with our previous studies, ANCOVA performed ondata collected at birth did not show overt detrimental effects of ges-tational CPF exposure on the number of delivered pups [Mean ±SEM: Veh = 11.417 ± 0.7; CPF = 8.455 ± 0.7, F(1,19) = 1.32,p = 0.262], the sex ratio [Mean ± SEM, Veh = 1.459 ± 0.3; CPF =1.91 ± 0.3, F(1,19) = 1.33, p = 0.26], and the overall litter weight[Mean ± SEM: Veh = 20.01 ± 0.81; CPF = 18.81 ± 1.15, F(1,19) =1.85, p = 0.19].

3.2. Social discrimination paradigm

Both Veh and CPFmice showed a progressive decline in social inves-tigation of the two stimulusmice throughout trials T1–T4 indicating ha-bituation regardless of treatment received [main effect of trialsF(3,117) = 19.95, G–G p b 0.001]. No preferences between social part-ners were observed, as mice of both treatment groups explored each ofthe two stimulus mice for a comparable amount of time which wasaround 50% of the total investigation time per trial. While no main

Table 1Social discrimination paradigm: total duration of wallrearing behavior during trials T1, T2, T3, and T4 in Vehand CPF-exposed mice of both sexes (M = males, F = -

females, main effect of sex, p b 0.001, see text for details).Data is expressed as means ± SEM.

Veh M 34.082 ± 5.959Veh F 11.232 ± 2.866CPF M 39.666 ± 8.736CPF F 17.505 ± 3.317

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treatment or sex effects were found, ANOVA yielded a significant inter-action between Treatment and Sex [F(1, 19)= 5.02, p= 0.0371]. Post-hoc comparisons showed that Veh females investigated significantlyless than Veh males (p b 0.05), while CPF females investigated signifi-cantly more than Veh females (p b 0.05) while not differing from CPFmales (Fig. 1).

In the social discrimination trial (T5), when one of the two familiarstimulusmicewas substituted at randomby a novel partner, allmice, ir-respective of the treatment, explored the novel social stimulus morethan the familiar one [F(1,39)= 35.29, p b 0.001], and no significant ef-fect of CPF, sex, or interaction between treatment and novelty responsewas evident (Fig. 1).

As regards spontaneous motor and exploratory behaviors record-ed throughout the test, mice expressed very low levels of bothgrooming and motor activity/immobility, but showed high levels ofwall rearing. Wall rearing was thus selected as an index of non-social activation (Table 1): the ANOVA showed a marked sex dimor-phism for this end point with males expressing significantly morewall rearing than females regardless of the prenatal treatment re-ceived (main effect of treatment, F(1,19) = 0.77, p = 0.3; main ef-fect of sex, F(1,19) = 19.66, p b 0.001).

3.3. Olfactory habituation/dishabituation test

The overall ANOVA including all odors (both non-social and social)indicated neither a main treatment effect nor a main effect of sex on ol-factory discrimination. A highly significant main effect of odors wasfound [F(4,144)= 45.76, G–Gp b 0.001] as the time spent in investigat-ing each non-social odor was significantly lower than that spent inves-tigating each social odor (ps b 0.01 after post-hoc comparisons). Thus,olfactory investigationwas analyzed separately for non-social and socialodors.

As for non-social odors, no main effect of sex or treatment wasshown. Amain effect of trial [F(2, 72)= 15.13, G–Gp b 0.001] indicatedsuccessful habituation to all the odors presented.Mice of both sexes andregardless of the treatment received spent a different amount of timesniffing the three odors [F(2, 72) = 5.58, G–G p = 0.021]. Significant

Fig. 1. Social discrimination paradigm— time spent in social investigation (Duration) of two stSocial investigation decreased from T1 to T4 in either sex regardless of treatment received (Trinvestigating the two stimulus mice averaged throughout the four trials. CPF females showedCPF males (interaction Treatment × Sex p = 0.037); # indicates a significant difference betwsubstituted one of the two familiar stimulus mice; ** indicates a significant difference betweemeans ± SEM. Females Veh, n = 9; CPF, n = 12; Males Veh, n = 10, CPF, n = 12.

habituation to each odor was observed as odor investigation in T1 wasalways greater than the investigation in T2 and T3 (ps b 0.01 afterpost hoc comparisons). Mice also showed a significant dishabituationwhen they were presented with a new odor, as seen in an increase intime spent sniffing the first swab of vanilla after water and the firstswab of almond after vanilla (ps b 0.05 after post hoc comparisons).

As for social odors, again no main effect of sex or treatment wasfound. ANOVA showed a significant main effect of Odor [F(1, 36) =5.86, p = 0.021] and Trials [F(2, 72) = 7.29, G–G p = 0.009] and asignificant two-way interaction between Odor and Sex [F(1, 36) =5.62, p = 0.023]. The interaction between Trial and Treatmentmissed statistical significance after Greenhouse–Geisser correction[F(2, 72) = 3.40, G–G p = 0.070]. As for the Odor × Sex interaction,post-hoc comparisons showed that females spent more time investi-gating the same sex than the opposite sex odor (p b 0.05), whilemales did not and were comparably interested in both kinds of socialodors. Unexpectedly, mice of either treatment and sex failed to showsignificant dishabituation to same-sex social odor, as only Veh ex-posed males increased the amount of time spent sniffing the firstswab of opposite-sex following presentation of the first swab ofsame-sex odor (T1 opposite-sex vs. T3 same-sex social odor). Differ-ent factors could account for such an apparent inability to discrimi-nate between the two different social odors; mice used in thisexperiment were sexually naïve, and it has been shown especiallyin out bred strains, that mating experience may be important in con-ferring attractiveness to the scent of a potential mate (Zinck and

imulus mice during trials T1, T2, T3, and T4 in Veh and CPF - exposed mice of both sexes.ial effect, p b 0.001, see Result Section, for details). The inset shows the mean time spenthigher levels of investigation than Veh females and were comparable to those of Veh andeen Veh females and both CPF females and Veh males (p b 0.05). In T5, a novel mousen investigation of the familiar vs novel stimulus mouse (p b 0.01). Data are expressed as

Fig. 2. Olfactory habituation/dishabituation test— A) Time spent sniffing a cotton swab soaked with non-social odors (water, vanilla extract, almond extract) displayed by Veh and CPF-exposed mice of both sexes during three repeated 1 min presentations (T1, T2, T3) of each odor. Veh and CPF mice of both sexes showed habituation and dishabituation to all the odorspresented. B) Time spent sniffing a cotton swab soakedwith twodifferent social odors (bedding of same sexmice or bedding of opposite sexmice) displayedbyVeh andCPF-exposedmiceduring three repeated 1minpresentations (T1, T2, T3) of the two odors. Females spent a higher proportion of time sniffing same sex odor than opposite sex odor. Nomain treatment or sexeffects were found. Data are expressed as means ± SEM. Females Veh, n = 16; CPF, n = 16; Males Veh, n = 14, CPF, n = 12.

36 A. De Felice et al. / Neurotoxicology and Teratology 46 (2014) 32–39

Lima, 2013). Additionally, neither experimental females nor scent-donor grouped females were in estrus at the time of testing; bothfactors could have reduced the interest of experimental subjects to-ward the opposite sex odor (Pankevich et al., 2004) (Fig. 2).

4. Discussion

The findings of the present study add further support to the hypoth-esis that CPF, one of the wider diffused insecticides of the OP class, sig-nificantly interferes with the development of sex dimorphic behaviorrepertoires in laboratory rodents. A main point emerges from thisstudy, namely that CPF administration during the last week of pregnan-cy abolished sex differences in mouse social investigative behavior andenhanced female offspring social investigation to the same levels foundin males without modifying responses to social novelty.

The effects of prenatal CPF exposure on female social behavior are inline with previous findings indicating enhanced responses to socialstimuli encountered in different experimental contexts. In more detail,our present data are in full agreement with findings obtained in femalemice exposed from GD 14 to 17 to 6 mg/kg/bw CPF, which showed amarked increase in social investigation andUSVemission during a socialrecognition test that recorded the investigative responses of the resi-dent female toward an intruder female placed in her home cage(Venerosi et al., 2006). Enhanced responsiveness to socially relevantcues has been confirmed in adult femalemice prenatally and neonatally

exposed to CPF in a very different experimental context; in the para-digm of maternal induction, where virgin females are presented withnew born pups in their home cage, CPF treated females showed signifi-cant enhancement of maternal care and pup-directed behavior (Ricceriet al., 2006).

In general despite having different task contingencies and require-ments, data collected so far indicate that CPF exposure causes pro-social activation and increased responsiveness/interest in social cues.Here we compared male and female CPF-exposed mice by applyingthe same social discrimination paradigm to the two sexes. This para-digm is made more complex by the presence of two social stimuli thatare repeatedly presented to the same animal before the final simulta-neous binary choice between a novel conspecific and the familiar one(Choleris et al., 2006). Similarly to other cognitive tests such as objector food recognition, this paradigm allows for the assessment of ananimal's capability to distinguish between two social stimuli, and thencompare the familiar individual with a novel one within the same test.We found that both CPF and control mice habituate to the two stimulusmice throughout the four habituation trials and successfully discrimi-nate the familiar mouse from the novel mouse in the final discrimina-tion trial, thus excluding the possibility of impaired habituation anddefective social novelty discrimination. Nonetheless, CPF treated fe-males displayed a much higher interest toward the stimulus micethan the control females and attained the level of social investigationcharacteristic of the male sex during the social habituation phase of

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the test. However, when challenged by the presentation of social novel-ty in T5, bothmales and females respondedwith increased investigationof the novel stimulus mouse regardless of the treatment received. Thus,somehow unexpectedly, themasculinizing effects of CPF in femalemiceappearedmore evident in a “basal” behavioralmeasure than in the pres-ence of an environmental challenge.

The masculinized profile was specific to social investigation as wallrearing duration, an index of markedly sex dimorphic motor activity,remained significantly lower than inmales in both CPF andVeh females.Notably, we observed a similar masculinization of female behaviorwhen assessing CD1 females exposed either in utero or neonatally toCPF at the same dose as the present study, in that CPF reduced anxietylevels in females making their behavior similar to that of males in an El-evated Plus Maze (Ricceri et al., 2006). Finally, in line with our findings,masculinization of behavior during radial maze performance (Levinet al., 2001) and locomotor activity habituation (Levin et al., 2002)was also a hallmark of prenatal and early neonatal CPF exposure in fe-male rats.

Several studies using behavioral paradigms similar to thoseemployed in the present study have reported sex differences in theduration of social investigation times, with males being significantlymore investigative than females (Markham and Juraska, 2007). Forexample, gonad-intact males spend more time investigating conspe-cifics than do gonad-intact females (Holmes et al., 2011) whereascastrated males and females exhibit similar low levels of investiga-tion toward conspecifics (Bluthe et al., 1993).

In this respect, testosterone treatment eliminated sex differences insocial investigation of a male conspecific, with females of the C57 strainexhibiting levels of investigation comparable to those of males (Tejadaand Rissman, 2012). Thus, sex differences in time spent investigatingconspecifics appear to be influenced by activation effects of hormones(Pierman et al., 2008).

We do not know the specific mechanism by which CPF elicits sex-selective effects on behavioral development. The range of behavioraleffects reported by different laboratories highlights a complex inter-action among sex-dependent susceptibility, exposure period, andtask requirement. However, the dampening of sex differences inworking memory, motor habituation (Levin et al., 2002), anxiety(Ricceri et al., 2006), and social investigation (present results) sup-ports the implication of hormonal factors and differentiation ofbrain regions involved in sexual dimorphisms. Interestingly, devel-opmental exposure to estrogenic pollutants such as bisphenol A(BPA) and methoxychlor (MXC) induced subtle behavioral alter-ations mainly in female mice, so that their exploratory behaviorwas more similar to control males' behavior than to the control fe-males' behavior (Gioiosa et al., 2013; Gioiosa et al., 2007). Exposureto BPA during development, besides reducing sex differences in re-sponse to novelty and exploration, also causes some alteration ofsexually dimorphic behaviors in CD1 mice by increasing male ag-gressive behavior at adulthood (Kawai et al., 2003) and altering ma-ternal responses (Palanza et al., 2002) with a range of effects similarto those reported for developmental CPF (Ricceri et al., 2006;Venerosi et al., 2006).

From amechanistic point of view, several data points to an action ofCPF on endocrine regulations of brain maturation and sexual differenti-ation. An inverse link between CPF exposure and reproductive hor-mones in humans has been reported (Meeker et al., 2006), anddifferent organophosphates including CPF significantly inhibit thehuman metabolism of steroid hormones in in vitro models (Hodgsonand Rose, 2006). We found that either in utero or postnatal CPF expo-sure decreased aromatase activity by 50% in the liver of neonatal miceat postnatal days 9 and 15; although this finding togetherwith the pres-ence of higher levels of the sex-specific Cyp2c activity at adulthood inmale mice suggests the occurrence of a long-lasting impairment in themetabolism of steroid hormones, the relevance of this effect at thebrain level has not been investigated yet (Buratti et al., 2011). At

doses comparable to those of the present work, a reduction in plasmalevels of testosterone and pituitary gonadotrophin hormones has beenreported in adult rats (Mandal and Das, 2012). In addition to evidencesuggesting an interference of CPF with steroid hormones, we haveshown that prenatal CPF has long lasting effects on neurohormones im-plicated in the modulation of social and affective responses such as OTandAVP as it increases OT and decreases AVP protein levels in the hypo-thalamus, particularly in the male sex (Tait et al., 2009). In this frame-work, we hypothesize that CPF modifies social behavior by acting onthe limbic/hypothalamic circuitries (markedly dimorphic in the twosexes) that modulate the appropriateness of social/investigative re-sponses. The involvement of estrogen receptors and OT in social recog-nition has been demonstrated by Choleris et al. (2003) through theanalysis of different strains of knockout female mice bearing deletionof ERα, ERβ, or OT genes. Notably, this same family of genes are alsomodified by gestational exposure to bisphenol A in parallel with chang-es in the response to social stimuli (Wolstenholme et al., 2012), suggest-ing a possible common mechanism by which different environmentalchemicals may increase the risk of sex-biased neurodevelopmental dis-orders affecting social functions.

Social investigation and recognition are mainly based on olfactoryfunction in rodents. In the olfactory habituation/dishabituation test,the olfactory cues were designed to measure the interest towardodors with and without social valence. In this respect, the result of theolfactory habituation/dishabituation test indicates preserved olfactoryabilities in CPF-treated mice and suggests normal function of both themain and accessory olfactory system in relation to the discriminationof the social valence of the odors presented (Baum and Keverne,2002). In more detail, results indicate that CPF treated mice of bothsexes are able to discriminate non-social from social odors, and, similar-ly to control mice, they showed amuch greater interest in social than innon-social odors. In addition, mice of both treatments and sexes allshowed normal levels of sniffing, habituation, and dishabituation tothe non-social odors, i.e. water, vanilla, and almond. A comparablyclear profile of habituation and dishabituation response was not foundfor social odor investigation, and this prevents us from drawing defini-tive conclusions on fine olfactory discrimination in CPF-exposed mice.Though CPF treated mice of both sexes appeared to habituate to socialodors to a lower extent than Veh-treated mice (possibly mirroring thesocial “arousal” described in this and previous studies from our labora-tory), a more thorough investigation of their olfactory investigative re-sponse should be performed, i.e. comparing social odors with differentdegrees of novelty, randomizing the sequence of presentation of the dif-ferent olfactory cues, and assessing olfactory preferences by presentinga binary choice between two different social odors with different sa-lience. Some methodological constraints accounting for the high vari-ability in individual response to social odors have been discussed inthe Results section. However, it is worth considering that most of stud-ies applying the olfactory habituation/dishabituation test used inbredstrains of mice where all individuals share the same genetically-determined scent signature (Hurst, 2009). The CD1 out-bred strain ismore similar to wild house mice as they are endowed with higher ge-netic variability that can result in a much higher individual variabilityin response to social odors. Finally, we did not observe a sexually dimor-phic profile in this test with the exception of a much greater interest insame-sex odor than in opposite-sex odor in females regardless of thegestational treatment received.

5. Conclusions

Altogether, the experimental findings reported here confirm and ex-tend previous evidence showing that in utero exposure to subtoxic dos-ages of CPF influences the maturation of sex-dimorphic clusters ofbehavioral items relevant for the proper expression of social responses.The finding that CPF, similarly to chemical compounds endowed withestrogen-like activity, blunts sex differences in diverse behavior

38 A. De Felice et al. / Neurotoxicology and Teratology 46 (2014) 32–39

patterns including learning, motor habituation, and social responsive-ness strengthens the hypothesis that this OP insecticide may behaveas a neuroendocrine disruptor at subtoxic doses (Venerosi et al.,2012). Evidence collected so far indicates that CPF targets multiple sig-naling systems that involve not only cholinergic mechanisms but alsosteroid hormones, hypothalamic neuropeptides, and serotonergictransmissions (Aldridge et al., 2005; Chen et al., 2011; Slotkin et al.,2009; Venerosi et al., 2010). The behavioral effects reported in experi-mental models might involve both central and peripheral mechanismswith the latter possibly implicating steroid-sensitive pathways and en-zymes that synthesize or degrade hormones.

To conclude, our findings add to previous evidence suggesting thatinterference with neuroendocrine functions might contribute to thedeleterious effects of early exposure to organophosphates in childrenby possibly increasing vulnerability to sex-biased neurodevelopmentaldisorders (Grandjean and Landrigan, 2014). Sex differences have beenbarely considered in clinical and epidemiological studies on behavioralCPF effects; the sex of the child was either not considered or notfound significant with the only exception being the study by Hortonet al. (2012) who found only a borderline significant interaction be-tween prenatal exposure to CPF and child sexwithmales beingmore af-fected than females in working memory scores. In a subgroup of thissame cohort, Rauh et al. (2012) recently found that high CPF exposurewas associated with brain anomalies in cortical thickness: notably, CPFchildren also displayed disruption of normal sexual dimorphisms insome brain structures and the expected sex differences were reversedin the high-CPF group. Under this perspective, future studies will needto focus on endocrine factors and/or sexually dimorphic features ofbrain maturation as determinants of the developmental neurotoxicityof CPF and other widely-diffused insecticides of the OP class.

Transparency Document

The Transparency Document associated with this article can befound, in the online version at doi: http://dx.doi.org/10.1016/j.ntt.2014.09.002.

Acknowledgments

This work was supported by the Italian Ministry of Health Grant,Young Researcher 2008, GR3-“Non-invasive tools for early detectionof Autism Spectrum Disorders”, and by ISS/Ministry of Health Grant5%3A “Role of neuroendocrine mechanisms in the etiology ofneurodevelopmental disorders”.

We acknowledge the help of Luigia Cancemi and Giovanni Dominiciin maintaining the mouse colony at the ISS.

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