Neurotoxicology and Teratology 46 (2014) 32–39

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Sex-dimorphic effects of gestational exposure to the organophosphate insecticide 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 b c

Section of Neurotoxicology and Neuroendocrinology, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00161 Rome, Italy Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00161 Rome, Italy

a r t i c l e

i n f o

Article history: Received 26 March 2014 Received in revised form 8 September 2014 Accepted 10 September 2014 Available online 28 September 2014 Keywords: Chlorpyrifos Social investigation Social discrimination Olfactory discrimination Sex differences Rodents

a b s t r a c t Several pieces of evidence from animal and human studies indicate that the organophosphate insecticide chlorpyrifos (CPF) acts as a developmental neurotoxicant at environmentally relevant doses, and it is possibly endowed with endocrine-disrupting activity. Data collected in rodent models show that developmental exposure to CPF at sub-toxic doses induces long-lasting and sex-dimorphic changes in social and investigative responses in exposed offspring. The aim of this study was to evaluate the effects of gestational CPF treatment on social and olfactory discrimination in adult mice of both sexes. Pregnant CD1 out-bred mice were exposed to CPF per os on gestational days (GD) 14–17 at the sub-toxic dose of 6 mg/kg/bw. At adulthood, male and female offspring underwent the same experimental paradigms, namely i) a social discrimination test where mice were presented with a simultaneous binary choice between a novel conspecific and a familiar one, and ii) an olfactory habituation/dishabituation test to evaluate their capability to discriminate between odors with different ecoethological salience (non-social vs. social odors). Results showed that in the social discrimination test prenatal CPF primarily affected the female sex by raising the investigation time in females to the same levels as found in vehicle- and CPF-exposed males. The ability to discriminate between a familiar and a novel social mate was not affected by CPF in either sex. In the olfactory habituation/dishabituation test, mice of both sexes successfully discriminated non-social from social odors regardless of the prenatal treatment received. These results confirm previous evidence indicating that developmental exposure to CPF causes long-lasting and sex-dimorphic changes in responsiveness to social cues, in the absence of significant impairment of social and olfactory discrimination capacity. These findings are discussed within the framework of recent data pointing to the limbic/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 organophosphate (OP) pesticide worldwide in both agricultural and urban communities. While the acute neurotoxicity of this chemical is associated with systemic and brain acetylcholinesterase (AChE) inhibition, an increasing body of experimental data suggests that, at low doses, CPF also targets non-cholinergic mechanisms (Eaton et al., 2008; Slotkin and Seidler, 2012).

⁎ Corresponding author at: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy. Tel.: +39 0649902106; fax: +39 064947821. E-mail addresses: [email protected] (A. De Felice), [email protected] (A. Venerosi), [email protected] (L. Ricceri), [email protected] (M. Sabbioni), [email protected] (M.L. Scattoni), fl[email protected] (F. Chiarotti), [email protected] (G. Calamandrei).

http://dx.doi.org/10.1016/j.ntt.2014.09.002 0892-0362/© 2014 Published by Elsevier Inc.

Several epidemiological studies to date support the hypothesis that at low, environmentally relevant doses, CPF acts as a developmental neurotoxicant. Analysis of longitudinal birth cohorts from either agricultural or urban communities found associations between prenatal exposure to CPF as measured in cord blood and a higher risk of mental and motor delay, pervasive developmental disorder (PDD), and hyperactive behaviors in exposed children (Rauh et al., 2006). The mechanisms by which CPF affect neurobehavioral development in exposed children are unclear, as are the possible delayed effects of this chemical at older ages. Studies in rodents demonstrated that at doses devoid of systemic toxicity, developmental exposure to CPF interferes with DNA synthesis, neuronal differentiation, synaptogenesis, and affects the expression levels of critical genes involved in brain development (Betancourt et al., 2006; Crumpton et al., 2000; Dam et al., 1998). Furthermore, fetal exposure to subtoxic doses of CPF alters neural systems beyond cholinergic transmission, such as serotonergic and dopaminergic neurotransmission, in a sex-dimorphic fashion (Slotkin and

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Seidler, 2007). Numerous rat studies have shown long-term effects on behavior after developmental exposure to subtoxic doses of CPF (Aldridge et al., 2004; Aldridge et al., 2005; Dam et al., 1999; Raines et al., 2001; Slotkin et al., 2002). Specifically, prenatal CPF exposure causes multiple behavioral alterations in rats tested from adolescence to adulthood; locomotor activity habituation and working and reference memory are affected with significant damping of sex differences in CPF exposed females (Levin et al., 2002). Similarly in the mouse species, CPF doses below the threshold for adverse effects on fetal growth or viability and maternal toxicity induce sex-dimorphic and long-term changes in a number of behavioral end points. In particular, in addition to increasing motor activity in the males, CPF targets different items of the social repertoire of laboratory mice in both sexes, including social recognition and pup-directed responses in virgin females (Ricceri et al., 2006), aggressive interactions between males to achieve social rank (Ricceri et al., 2006), social recognition (Venerosi et al., 2006), and nest defense response in lactating females (Venerosi et al., 2009). We have also shown that CPF has long lasting effects on the neurohormones implicated in the modulation of social and affective responses such as oxytocin (OT) and vasopressin (AVP) (Tait et al., 2009). Notably, hypothalamic neuropeptides are considered components of a sex-dimorphic hypothalamic–limbic micronet modulating social responses in rodents (Choleris et al., 2003), and regulating social recognition at the level of the olfactory system in rodents (Bielsky and Young, 2004; Wacker and Ludwig, 2012). On the basis of this experimental evidence, we hypothesized that one of the possible mechanisms underlying CPF behavioral toxicity may be the interference with endocrine factors and/or sexually dimorphic features of brain maturation (Venerosi et al., 2012). As social investigation and discrimination are markedly sex-dimorphic in rodents, we expect to find sex-dependent vulnerability to CPF effects by assessing adult mice of both sexes with the same experimental paradigm. Specifically in the present study, we evaluated the effects of gestational CPF exposure on social investigation and discrimination by applying a social discrimination test where two social stimuli are repeatedly presented to the same animal before the final simultaneous binary choice between a novel conspecific and a familiar one (Choleris et al., 2003). Additionally, in order to verify the possibility that CPF alters social responses by interfering with the detection and/or processing of olfactory cues, we assessed the same mice in a habituation/dishabituation olfactory test where non-social (water, vanilla, almond) or social (same-sex urine, opposite-sex urine) odors were presented sequentially to mice to evaluate their ability to distinguish between odors with different ethological value and to habituate to them (Yang and Crawley, 2009). CPF or its vehicle were administered at the sub-toxic dose of 6 mg/kg/bw by oral gavage from gestational day (GD) 14 to 17 to pregnant mice of the CD1 strain.

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2. Materials and methods

gestational phases (for doses comprised between 1 and 5 mg/kg/bw) for the effects of prenatal CPF exposure on both neural systems and behavior (Aldridge et al., 2005; Qiao et al., 2003). This same late gestation exposure window and the dose used in our present study was found to produce significant effects on adult social responses and hypothalamic neuropeptide levels in mice (Ricceri et al., 2006; Tait et al., 2009). Notably, the lowest observed adverse effect level (LOAEL) established by the EU for neurotoxicity in rats after chronic exposure is 10 mg/kg/bw/day, although most of the developmental neurotoxicity studies in rats and mice found delayed behavioral effects at doses comprised between 1 and 6 mg/kg/bw/day. Estimated CPF exposure in the general population including children and women of childbearing age today is primarily through diet and in the 10−1 to 10−3 μg/kg-d dose range (Li et al., 2012), although much higher exposure 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 performance of treated dams (pregnancy length, number of pups at delivery, sex ratio), and it does not induce overt toxic symptoms in dams or major effects on pups' health parameters such as weight at delivery and impairment of growth rate (Ricceri et al., 2006). Following CPF dose administration to pregnant mice after applying the same treatment schedule of the present study, we found no effects on brain AChE activity and a mild transient inhibition (20% of control values) in serum AChE activity in offspring when measured at birth 24 h after the last exposure (Ricceri et al., 2006). We cannot exclude that limited but significant brain AChE inhibition could have been present at earlier time points and recovery could have occurred by 24 h following exposure as previously indicated in the rat species by (Mattsson et al., 2000). Of the twenty-six pregnant females treated, four females (two Veh and two CPF treated) gave birth on GD 17 and were thus excluded from further study. A total of twenty-two litters (12 Vehicle-treated and 10 CPF-treated) were used. On the day of birth, number of pups delivered, overall weight of the litter, and sex ratio were recorded to verify the absence of CPF effects on reproductive performance by considering the body weight of each pregnant female before the beginning of treatment as a covariate. We assessed the sex of the pups by evaluation of ano-genital distance and litters were culled to a maximum of 10 pups while always trying to maintain a comparable number of pups of the two sexes within the litter (0.6 ≤ sex ratio ≤ 1.5). Offspring were weaned on postnatal day 23, housed in cages containing littermates of the same sex, and left undisturbed until the beginning of the behavioral assessment. At adulthood, one female and one male 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 litters were assessed in the olfactory habituation/dishabituation test. All experiments on animals were performed according to the European Community Council Directive 2010/63/EU and to Italian Legislation on Animal Experimentation (Legislative Decree 116/92).

2.1. Subjects and treatment

2.2. Social discrimination paradigm

Forty male and female mice of the out bred Swiss-derived strain (CD1, Harlan, S. Pietro al Natisone, Italy), were housed in pairs in breeding cages (polycarbonate cages 33 × 13 × 14 cm) with a 12-h light–dark cycle (lights on at 8 pm) with free access to food (enrichment standard diet 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 discovery of the vaginal plug. On GD14, 26 pregnant females were randomly assigned 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 of 0.1 ml/10 g at a dose of 6 mg/kg/bw) or its vehicle was administered to pregnant females from GD 14 to 17 by intraoral gavages. Extensive work by Slotkin's group in rats has shown a greater sensitivity of late

On postnatal day 70, both females (Veh, n = 12; CPF, n = 9; one female was excluded due to loss of video recorded data) and males (Veh, n = 12; CPF, n = 10) from each treatment underwent a social discrimination test as described by Choleris et al. (2006). The test was performed during the dark phase of the light/dark cycle in a novel polycarbonate cage (48 × 27 × 21 cm) where the experimental subject underwent a five trial social discrimination test between two social stimuli (age- and sex-matched CD1 mice). In each trial (T1–T5), two wired cylinders (Galaxy Cup, Kitchen Plus, http://www.kitchen-plus.com; diameter 10 cm, height 10.5 cm), each containing a stimulus mouse from different cages, were placed in two opposite sides of the test cage. The use of wired cylinders allowed for passage of olfactory cues while preventing direct interactions between stimulus and experimental mice. This ensured that experimental mice

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did not deposit their own scent onto stimulus mice, while still eliciting high social interest and investigation from experimental mice. Each trial lasted 5 min. In trials T1–T4, the same two stimulus mice were placed in identical locations within the experimental cage, whereas in trial T5 one of the two stimulus mice was novel while the other was the 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 the experimental subjects remained in the test cage in the presence of the two empty cylinders used during T1–T5 trials. Importantly, in all tests the location of stimulus mice was kept constant to make spatial cues irrelevant to the task. Two procedures were adopted to limit bias due to novelty and stress effects: i) before testing, the experimental subjects were left undisturbed for one hour in the experimental cage with two empty wired cylinders to habituate to them; ii) stimulus mice were habituated to cylinders several days prior to the day of the test until they spontaneously 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-social responses to the stimulus mice. To this aim, trials (T1–T5) were video recorded and observations were analyzed using the Observer Video Analysis software (Noldus Information Technology, Wageningen, the Netherlands) by a trained experimenter blind to the treatment received by each mouse. The time spent in social investigation (sniffing every body part of the stimulus mice through the wired cylinders) was scored separately on each of the five trials. In T1–T4 where the familiarity of the mice at each trial was equivalent, animals were expected to investigate each stimulus mouse around 50% of the total time (random choice) showing a similar habituation profile to both stimulus mice; while in T5, if the mice can successfully discriminate between the novel and the familiar stimulus mouse, the proportion of time spent investigating the novel stimulus mouse should be significantly higher than that spent in investigating the familiar mouse. Thus, as test conditions changed markedly between T1–T4 and T5 sessions, data were analyzed separately 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 five 5 min-trials.

2.3. Olfactory habituation/dishabituation The olfactory habituation/dishabituation test analyzes the animal's tendency to investigate novel smells and the mouse's ability to detect and differentiate odors with different eco-ethological values. The experimental paradigm applied here is a slightly modified version of the procedure described by Yang and Crawley (2009). A total of thirty-two females (Veh, n = 16; CPF, n = 16) and twentysix males (Veh, n = 14; CPF, n = 12) were used for this test. The test was performed during the dark phase of the light/dark cycle in a polycarbonate cage (33 × 13 × 14 cm) identical to the home cage but with clean sawdust, whereas the experimental subject was exposed to a cotton swab laterally introduced through the cage lid. Each mouse was introduced to the experimental cage for 1 h prior to testing to habituate them to the setting. Females were tested when not in estrous as assessed by visual observation of the vagina (Byers et al., 2012). The test was characterized by sequential exposure to different non-social and social odors. Each trial lasted 2 min, and each odor was presented in a consecutive 3-trial (T1, T2, and T3) sequence. Sequences of three identical swabs determine habituation to the same odor, while switching to another odor determine dishabituation. Habituation was defined as the decrease from trial 1 to trial 3 in time spent sniffing the cotton swab soaked with a given non-social or social odor. Dishabituation was defined as the increase in time spent sniffing the cotton swab soaked with a novel odor, following three successive presentations of the previous social or non-social odor.

Odor presentation was performed by inserting into the cage a cotton swab 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 cotton swab in a zigzag pattern across the bottom surface of a cage of unfamiliar mice of d) same strain, same sex; e) same strain, different sex. Social odors were collected from one-week-old bedding of either grouphoused females or individually-housed males. The order of swab presentation was: water 1, water 2, water 3, vanilla 1, vanilla 2, vanilla 3, almond 1, almond 2, almond 3, social odor same sex 1, social odor same sex 2, social odor same sex 3, social odor different sex 1, social odor different sex 2, social odor different sex 3. Time spent sniffing each odor was scored when the animal oriented toward the cotton tip with its nose being within 2 cm of the cotton swab. 2.4. Statistical analysis Reproductive performance data were analyzed by analysis of covariance (ANCOVA) with treatment as the between-subject fixed factor (2 levels), and body weight of pregnant females before the beginning of treatment as the covariate. Behavioral data from the social discrimination test during trials (T1–T4) were analyzed by analysis of variance (ANOVA) with prenatal treatment (2 levels) as the between-litter fixed factor, sex (2 levels) as the within-litter fixed factor, and trials as the repeated measures factor (4 levels). Social investigation in T5 was analyzed separately applying the ANOVA model with prenatal treatment (2 levels) as the between-litter fixed factor, sex (2 levels) as the within-litter fixed factor, and familiarity/novelty (time spent in investigating either the familiar or the novel stimulus mouse; 2 levels) as the within-subject fixed factor. In the olfactory habituation/dishabituation paradigm, more than 1 female and 1 male were used per litter. Additional animals were selected randomly and the litter effect was taken into account for calculating the mean square of errors in the ANOVA. Time sniffing the cotton swab soaked with different non-social and social odors was analyzed by the ANOVA model with prenatal treatment (2 levels) as the betweenlitter fixed factor, sex (2 levels) as the within-litter fixed factor, and odors (5 levels) and trials (3 levels) as the repeated measures factors. Greenhouse–Geisser correction (G–G) was used to deal with violation of the sphericity assumption when testing repeated measures factors. Multiple comparisons were performed using Tukey's test that can also be used in the absence of a significant (p b 0.05) interaction to minimize both Type I and Type II errors. 3. Results 3.1. Reproductive performances In agreement with our previous studies, ANCOVA performed on data collected at birth did not show overt detrimental effects of gestational 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 CPF mice showed a progressive decline in social investigation of the two stimulus mice throughout trials T1–T4 indicating habituation regardless of treatment received [main effect of trials F(3,117) = 19.95, G–G p b 0.001]. No preferences between social partners were observed, as mice of both treatment groups explored each of the two stimulus mice for a comparable amount of time which was around 50% of the total investigation time per trial. While no main

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treatment or sex effects were found, ANOVA yielded a significant interaction between Treatment and Sex [F(1, 19) = 5.02, p = 0.0371]. Posthoc comparisons showed that Veh females investigated significantly less than Veh males (p b 0.05), while CPF females investigated significantly more than Veh females (p b 0.05) while not differing from CPF males (Fig. 1). In the social discrimination trial (T5), when one of the two familiar stimulus mice was substituted at random by a novel partner, all mice, irrespective of the treatment, explored the novel social stimulus more than the familiar one [F(1,39) = 35.29, p b 0.001], and no significant effect of CPF, sex, or interaction between treatment and novelty response was evident (Fig. 1). As regards spontaneous motor and exploratory behaviors recorded throughout the test, mice expressed very low levels of both grooming and motor activity/immobility, but showed high levels of wall rearing. Wall rearing was thus selected as an index of nonsocial activation (Table 1): the ANOVA showed a marked sex dimorphism for this end point with males expressing significantly more wall rearing than females regardless of the prenatal treatment received (main effect of treatment, F(1,19) = 0.77, p = 0.3; main effect 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 olfactory discrimination. A highly significant main effect of odors was found [F(4,144) = 45.76, G–G p b 0.001] as the time spent in investigating each non-social odor was significantly lower than that spent investigating each social odor (ps b 0.01 after post-hoc comparisons). Thus, olfactory investigation was analyzed separately for non-social and social odors. As for non-social odors, no main effect of sex or treatment was shown. A main effect of trial [F(2, 72) = 15.13, G–G p b 0.001] indicated successful habituation to all the odors presented. Mice of both sexes and regardless of the treatment received spent a different amount of time sniffing the three odors [F(2, 72) = 5.58, G–G p = 0.021]. Significant

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Table 1 Social discrimination paradigm: total duration of wall rearing behavior during trials T1, T2, T3, and T4 in Veh and 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 Veh F CPF M CPF F

34.082 11.232 39.666 17.505

± ± ± ±

5.959 2.866 8.736 3.317

habituation to each odor was observed as odor investigation in T1 was always greater than the investigation in T2 and T3 (ps b 0.01 after post hoc comparisons). Mice also showed a significant dishabituation when they were presented with a new odor, as seen in an increase in time spent sniffing the first swab of vanilla after water and the first swab of almond after vanilla (ps b 0.05 after post hoc comparisons). As for social odors, again no main effect of sex or treatment was found. 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 a significant two-way interaction between Odor and Sex [F(1, 36) = 5.62, p = 0.023]. The interaction between Trial and Treatment missed 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 investigating the same sex than the opposite sex odor (p b 0.05), while males did not and were comparably interested in both kinds of social odors. Unexpectedly, mice of either treatment and sex failed to show significant dishabituation to same-sex social odor, as only Veh exposed males increased the amount of time spent sniffing the first swab of opposite-sex following presentation of the first swab of same-sex odor (T1 opposite-sex vs. T3 same-sex social odor). Different factors could account for such an apparent inability to discriminate between the two different social odors; mice used in this experiment were sexually naïve, and it has been shown especially in out bred strains, that mating experience may be important in conferring attractiveness to the scent of a potential mate (Zinck and

Fig. 1. Social discrimination paradigm — time spent in social investigation (Duration) of two stimulus mice during trials T1, T2, T3, and T4 in Veh and CPF - exposed mice of both sexes. Social investigation decreased from T1 to T4 in either sex regardless of treatment received (Trial effect, p b 0.001, see Result Section, for details). The inset shows the mean time spent investigating the two stimulus mice averaged throughout the four trials. CPF females showed higher levels of investigation than Veh females and were comparable to those of Veh and CPF males (interaction Treatment × Sex p = 0.037); # indicates a significant difference between Veh females and both CPF females and Veh males (p b 0.05). In T5, a novel mouse substituted one of the two familiar stimulus mice; ** indicates a significant difference between investigation of the familiar vs novel stimulus mouse (p b 0.01). Data are expressed as means ± SEM. Females Veh, n = 9; CPF, n = 12; Males Veh, n = 10, CPF, n = 12.

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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 CPFexposed 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 odors presented. B) Time spent sniffing a cotton swab soaked with two different social odors (bedding of same sex mice or bedding of opposite sex mice) displayed by Veh and CPF-exposed mice during three repeated 1 min presentations (T1, T2, T3) of the two odors. Females spent a higher proportion of time sniffing same sex odor than opposite sex odor. No main treatment or sex effects were found. Data are expressed as means ± SEM. Females Veh, n = 16; CPF, n = 16; Males Veh, n = 14, CPF, n = 12.

Lima, 2013). Additionally, neither experimental females nor scentdonor grouped females were in estrus at the time of testing; both factors could have reduced the interest of experimental subjects toward the opposite sex odor (Pankevich et al., 2004) (Fig. 2). 4. Discussion The findings of the present study add further support to the hypothesis that CPF, one of the wider diffused insecticides of the OP class, significantly interferes with the development of sex dimorphic behavior repertoires in laboratory rodents. A main point emerges from this study, namely that CPF administration during the last week of pregnancy abolished sex differences in mouse social investigative behavior and enhanced female offspring social investigation to the same levels found in males without modifying responses to social novelty. The effects of prenatal CPF exposure on female social behavior are in line with previous findings indicating enhanced responses to social stimuli encountered in different experimental contexts. In more detail, our present data are in full agreement with findings obtained in female mice exposed from GD 14 to 17 to 6 mg/kg/bw CPF, which showed a marked increase in social investigation and USV emission during a social recognition test that recorded the investigative responses of the resident female toward an intruder female placed in her home cage (Venerosi et al., 2006). Enhanced responsiveness to socially relevant cues has been confirmed in adult female mice prenatally and neonatally

exposed to CPF in a very different experimental context; in the paradigm of maternal induction, where virgin females are presented with new born pups in their home cage, CPF treated females showed significant enhancement of maternal care and pup-directed behavior (Ricceri et al., 2006). In general despite having different task contingencies and requirements, data collected so far indicate that CPF exposure causes prosocial activation and increased responsiveness/interest in social cues. Here we compared male and female CPF-exposed mice by applying the same social discrimination paradigm to the two sexes. This paradigm is made more complex by the presence of two social stimuli that are repeatedly presented to the same animal before the final simultaneous binary choice between a novel conspecific and the familiar one (Choleris et al., 2006). Similarly to other cognitive tests such as object or food recognition, this paradigm allows for the assessment of an animal's capability to distinguish between two social stimuli, and then compare the familiar individual with a novel one within the same test. We found that both CPF and control mice habituate to the two stimulus mice throughout the four habituation trials and successfully discriminate the familiar mouse from the novel mouse in the final discrimination trial, thus excluding the possibility of impaired habituation and defective social novelty discrimination. Nonetheless, CPF treated females displayed a much higher interest toward the stimulus mice than the control females and attained the level of social investigation characteristic of the male sex during the social habituation phase of

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the test. However, when challenged by the presentation of social novelty in T5, both males and females responded with increased investigation of the novel stimulus mouse regardless of the treatment received. Thus, somehow unexpectedly, the masculinizing effects of CPF in female mice appeared more evident in a “basal” behavioral measure than in the presence of an environmental challenge. The masculinized profile was specific to social investigation as wall rearing duration, an index of markedly sex dimorphic motor activity, remained significantly lower than in males in both CPF and Veh females. Notably, we observed a similar masculinization of female behavior when assessing CD1 females exposed either in utero or neonatally to CPF at the same dose as the present study, in that CPF reduced anxiety levels in females making their behavior similar to that of males in an Elevated Plus Maze (Ricceri et al., 2006). Finally, in line with our findings, masculinization of behavior during radial maze performance (Levin et al., 2001) and locomotor activity habituation (Levin et al., 2002) was also a hallmark of prenatal and early neonatal CPF exposure in female rats. Several studies using behavioral paradigms similar to those employed in the present study have reported sex differences in the duration of social investigation times, with males being significantly more investigative than females (Markham and Juraska, 2007). For example, gonad-intact males spend more time investigating conspecifics than do gonad-intact females (Holmes et al., 2011) whereas castrated males and females exhibit similar low levels of investigation toward conspecifics (Bluthe et al., 1993). In this respect, testosterone treatment eliminated sex differences in social investigation of a male conspecific, with females of the C57 strain exhibiting levels of investigation comparable to those of males (Tejada and Rissman, 2012). Thus, sex differences in time spent investigating conspecifics appear to be influenced by activation effects of hormones (Pierman et al., 2008). We do not know the specific mechanism by which CPF elicits sexselective effects on behavioral development. The range of behavioral effects reported by different laboratories highlights a complex interaction among sex-dependent susceptibility, exposure period, and task requirement. However, the dampening of sex differences in working memory, motor habituation (Levin et al., 2002), anxiety (Ricceri et al., 2006), and social investigation (present results) supports the implication of hormonal factors and differentiation of brain regions involved in sexual dimorphisms. Interestingly, developmental exposure to estrogenic pollutants such as bisphenol A (BPA) and methoxychlor (MXC) induced subtle behavioral alterations mainly in female mice, so that their exploratory behavior was more similar to control males' behavior than to the control females' behavior (Gioiosa et al., 2013; Gioiosa et al., 2007). Exposure to BPA during development, besides reducing sex differences in response to novelty and exploration, also causes some alteration of sexually dimorphic behaviors in CD1 mice by increasing male aggressive behavior at adulthood (Kawai et al., 2003) and altering maternal responses (Palanza et al., 2002) with a range of effects similar to those reported for developmental CPF (Ricceri et al., 2006; Venerosi et al., 2006). From a mechanistic point of view, several data points to an action of CPF on endocrine regulations of brain maturation and sexual differentiation. An inverse link between CPF exposure and reproductive hormones in humans has been reported (Meeker et al., 2006), and different organophosphates including CPF significantly inhibit the human metabolism of steroid hormones in in vitro models (Hodgson and Rose, 2006). We found that either in utero or postnatal CPF exposure decreased aromatase activity by 50% in the liver of neonatal mice at postnatal days 9 and 15; although this finding together with the presence of higher levels of the sex-specific Cyp2c activity at adulthood in male mice suggests the occurrence of a long-lasting impairment in the metabolism of steroid hormones, the relevance of this effect at the brain level has not been investigated yet (Buratti et al., 2011). At

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doses comparable to those of the present work, a reduction in plasma levels of testosterone and pituitary gonadotrophin hormones has been reported in adult rats (Mandal and Das, 2012). In addition to evidence suggesting an interference of CPF with steroid hormones, we have shown that prenatal CPF has long lasting effects on neurohormones implicated in the modulation of social and affective responses such as OT and AVP as it increases OT and decreases AVP protein levels in the hypothalamus, particularly in the male sex (Tait et al., 2009). In this framework, we hypothesize that CPF modifies social behavior by acting on the limbic/hypothalamic circuitries (markedly dimorphic in the two sexes) that modulate the appropriateness of social/investigative responses. The involvement of estrogen receptors and OT in social recognition has been demonstrated by Choleris et al. (2003) through the analysis of different strains of knockout female mice bearing deletion of ERα, ERβ, or OT genes. Notably, this same family of genes are also modified by gestational exposure to bisphenol A in parallel with changes in the response to social stimuli (Wolstenholme et al., 2012), suggesting a possible common mechanism by which different environmental chemicals may increase the risk of sex-biased neurodevelopmental disorders affecting social functions. Social investigation and recognition are mainly based on olfactory function in rodents. In the olfactory habituation/dishabituation test, the olfactory cues were designed to measure the interest toward odors with and without social valence. In this respect, the result of the olfactory habituation/dishabituation test indicates preserved olfactory abilities in CPF-treated mice and suggests normal function of both the main and accessory olfactory system in relation to the discrimination of the social valence of the odors presented (Baum and Keverne, 2002). In more detail, results indicate that CPF treated mice of both sexes are able to discriminate non-social from social odors, and, similarly to control mice, they showed a much greater interest in social than in non-social odors. In addition, mice of both treatments and sexes all showed normal levels of sniffing, habituation, and dishabituation to the non-social odors, i.e. water, vanilla, and almond. A comparably clear profile of habituation and dishabituation response was not found for social odor investigation, and this prevents us from drawing definitive conclusions on fine olfactory discrimination in CPF-exposed mice. Though CPF treated mice of both sexes appeared to habituate to social odors to a lower extent than Veh-treated mice (possibly mirroring the social “arousal” described in this and previous studies from our laboratory), a more thorough investigation of their olfactory investigative response should be performed, i.e. comparing social odors with different degrees of novelty, randomizing the sequence of presentation of the different olfactory cues, and assessing olfactory preferences by presenting a binary choice between two different social odors with different salience. Some methodological constraints accounting for the high variability in individual response to social odors have been discussed in the Results section. However, it is worth considering that most of studies applying the olfactory habituation/dishabituation test used inbred strains of mice where all individuals share the same geneticallydetermined scent signature (Hurst, 2009). The CD1 out-bred strain is more similar to wild house mice as they are endowed with higher genetic variability that can result in a much higher individual variability in response to social odors. Finally, we did not observe a sexually dimorphic profile in this test with the exception of a much greater interest in same-sex odor than in opposite-sex odor in females regardless of the gestational treatment received. 5. Conclusions Altogether, the experimental findings reported here confirm and extend previous evidence showing that in utero exposure to subtoxic dosages of CPF influences the maturation of sex-dimorphic clusters of behavioral items relevant for the proper expression of social responses. The finding that CPF, similarly to chemical compounds endowed with estrogen-like activity, blunts sex differences in diverse behavior

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patterns including learning, motor habituation, and social responsiveness strengthens the hypothesis that this OP insecticide may behave as a neuroendocrine disruptor at subtoxic doses (Venerosi et al., 2012). Evidence collected so far indicates that CPF targets multiple signaling systems that involve not only cholinergic mechanisms but also steroid hormones, hypothalamic neuropeptides, and serotonergic transmissions (Aldridge et al., 2005; Chen et al., 2011; Slotkin et al., 2009; Venerosi et al., 2010). The behavioral effects reported in experimental models might involve both central and peripheral mechanisms with the latter possibly implicating steroid-sensitive pathways and enzymes that synthesize or degrade hormones. To conclude, our findings add to previous evidence suggesting that interference with neuroendocrine functions might contribute to the deleterious effects of early exposure to organophosphates in children by possibly increasing vulnerability to sex-biased neurodevelopmental disorders (Grandjean and Landrigan, 2014). Sex differences have been barely considered in clinical and epidemiological studies on behavioral CPF effects; the sex of the child was either not considered or not found significant with the only exception being the study by Horton et al. (2012) who found only a borderline significant interaction between prenatal exposure to CPF and child sex with males being more affected than females in working memory scores. In a subgroup of this same cohort, Rauh et al. (2012) recently found that high CPF exposure was associated with brain anomalies in cortical thickness: notably, CPF children also displayed disruption of normal sexual dimorphisms in some brain structures and the expected sex differences were reversed in the high-CPF group. Under this perspective, future studies will need to focus on endocrine factors and/or sexually dimorphic features of brain maturation as determinants of the developmental neurotoxicity of CPF and other widely-diffused insecticides of the OP class. Transparency Document The Transparency Document associated with this article can be found, 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 detection of Autism Spectrum Disorders”, and by ISS/Ministry of Health Grant 5%3A “Role of neuroendocrine mechanisms in the etiology of neurodevelopmental disorders”. We acknowledge the help of Luigia Cancemi and Giovanni Dominici in maintaining the mouse colony at the ISS. References Aldridge JE, Seidler FJ, Slotkin TA. Developmental exposure to chlorpyrifos elicits sexselective alterations of serotonergic synaptic function in adulthood: critical periods and regional selectivity for effects on the serotonin transporter, receptor subtypes, and cell signaling. Environ Health Perspect 2004;112:148–55. Aldridge JE, Meyer A, Seidler FJ, Slotkin TA. Alterations in central nervous system serotonergic and dopaminergic synaptic activity in adulthood after prenatal or neonatal chlorpyrifos exposure. Environ Health Perspect 2005;113:1027–31. Baum MJ, Keverne EB. Sex difference in attraction thresholds for volatile odors from male and estrous female mouse urine. Horm Behav 2002;41:213–9. Betancourt AM, Burgess SC, Carr RL. Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain. Toxicol Sci 2006;92:500–6. Bielsky IF, Young LJ. Oxytocin, vasopressin, and social recognition in mammals. Peptides 2004;25:1565–74. Bluthe RM, Gheusi G, Dantzer R. Gonadal steroids influence the involvement of arginine vasopressin in social recognition in mice. Psychoneuroendocrinology 1993;18: 323–35. Buratti FM, De Angelis G, Ricceri L, Venerosi A, Calamandrei G, Testai E. Foetal and neonatal exposure to chlorpyrifos: biochemical and metabolic alterations in the mouse liver at different developmental stages. Toxicology 2011;280:98–108. Byers SL, Wiles MV, Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One 2012;7:e35538.

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