Perinatal 17α-ethinylestradiol exposure affects formalin-induced responses in middle-aged male (but not female) rats

Hormones and Behavior 73 (2015) 116–124

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Perinatal 17α-ethinylestradiol exposure affects formalin-induced responses in middle-aged male (but not female) rats Ilaria Ceccarelli, Paolo Fiorenzani, Daniele Della Seta, Anna Maria Aloisi ⁎ Department of Medicine, Surgery and Neuroscience, University of Siena, via Aldo Moro, 2, 53100 Siena, Italy

a r t i c l e

i n f o

Article history: Received 12 December 2014 Revised 16 June 2015 Accepted 1 July 2015 Available online 6 July 2015 Keywords: Xenoestrogens Formalin test Pain Developmental exposure Sex differences Gonadal hormones

a b s t r a c t 17α-Ethinylestradiol (EE), the main component of the contraceptive pill, is a synthetic estrogen found in rivers of the United States and Europe as an environmental contaminant. It is one of the most studied xenoestrogens due to its possible effect on the reproductive system. In the present study we evaluated the modulation of pain responses induced by formalin injection (licking, flexing, paw-jerk) in 8-month-old male and female offspring of female rats treated with two different doses of EE (4 ng/kg/day or 400 ng/kg/day) during pregnancy and lactation. Spontaneous behaviors and gonadal hormone levels were also determined. Both concentrations of EE induced an increase of pain behaviors in males only, i.e. higher flexing and licking of the formalin-injected paw than in OIL-exposed rats, during the second, inflammatory, phase of the formalin test. Grooming duration was increased by EE exposure in both males and females. Prenatal EE exposure (both concentrations) decreased estradiol plasma levels in the formalin-injected females but not in the males. These results underline the possibility that exposure to an environmental contaminant during the critical period of development can affect neural processes (such as those involved in pain modulation) during adulthood, indicating long-term changes in brain circuitry. However, such changes may be different in males and females. © 2015 Elsevier Inc. All rights reserved.

Introduction Estrogens have a wide range of actions in the human body. They have profound effects on sexual differentiation but also modulate cardiovascular function, bone condition, hemostasis, water and salt balance, and the metabolic rate. “Xenoestrogens” is the name given to disrupting chemicals able to mimic the action of endogenous estrogens (Fisher, 2004). These compounds enter the environment from different sources and can be synthetic estrogen-like chemicals like bisphenol A (BPA) or synthetic estrogens used as drugs. Classically, xenoestrogens exert their biological effects by binding to nuclear estrogen receptors (ERs) which function as transcription factors (Dang et al., 2009; Thayer et al., 2001; Timms et al., 2005). Recent studies have documented the complexity and detail of estrogen and xenoestrogen functions in the central nervous system (CNS) and have shown their importance in many cardinal brain systems (Hughes et al., 2009; Remage-Healey, 2014; vom Saal and Hughes, 2005); importantly, several studies have reported alterations of cognitive functions in humans and laboratory animals following xenoestrogen exposure (Colborn et al., 1993; Jacobson and Jacobson, 1996; Lephart et al., 2004; Ryan and Vanderbergh, 2006; Welshons et al., 2003). ⁎ Corresponding author at: Department of Medicine, Surgery and Neuroscience, University of Siena, via Aldo Moro, 2, 53100 Siena, Italy. E-mail address: [email protected] (A.M. Aloisi).

http://dx.doi.org/10.1016/j.yhbeh.2015.07.001 0018-506X/© 2015 Elsevier Inc. All rights reserved.

The nociceptive system is strongly modulated by estrogens at both the spinal and supraspinal levels via their binding to ER-α and ER-ß (as well as GPR30), eliciting both genomic and non-genomic effects (Chaban and Micevych, 2002; Ceccarelli et al., 2003; Gaumond et al., 2005; Xu et al., 2008; Dun et al., 2009). Recent studies suggest that estrogens may be involved in peripheral pain signal transduction, both up-regulating P2X3 expression (Ma et al., 2011) and modulating the downstream cAMP-PKA-ERK1/2 signal pathway following their binding to ER-α and GPR30 (Lu et al., 2013). Moreover several supraspinal pain circuits are modulated by estrogens (Amandusson and Blomqvist, 2013). In a previous experiment we demonstrated that intracerebroventricular (i.c.v.) injection of 17-β estradiol in intact and gonadectomized male rats increased the more centrally integrated formalininduced pain response (licking of the injected paw) during the second phase of the formalin test and that this increase was counteracted by naloxone (Aloisi and Ceccarelli, 2000), ICI 182–780 (ER-α antagonist) and β-funaltrexamine (μ-opioid receptor antagonist) administration in intact male rats (Ceccarelli et al., 2004). Epidemiological and animal studies have clearly shown that xenoestrogens taken during development have disruptive effects not only on reproductive organs but also on the brain (Dugard et al., 2001; Ferguson et al., 2014; Frye et al., 2012). The most critical time for the action of xenoestrogens is the perinatal period (in rodents from the prenatal period to 2 weeks of age) during which the brain actively develops through the expression of many genes under the influence of a variety of hormones. In this way the effect of xenoestrogen exposure will

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appear later as behavioral changes such as deficits of emotional, reproductive or learning behaviors or changes in sensory functions. 17α-Ethinylestradiol (EE) is a pure synthetic estrogen and the main estrogenic component of the contraceptive pill. EE binds to estrogen receptors, in particular ERα, with much higher affinity than endogenous estradiol (Blair et al., 2000; Ferguson et al., 2003). It is estimated that each year almost 2 million women using the contraceptive pill become pregnant and unintentional exposure of the developing human fetus to EE can occur if oral contraception is continued through the early part of undetected pregnancy (Li et al., 1995). To mimic this condition we administered a high dose of EE (as taken with a contraceptive pill) to dams during pregnancy or during lactation which resulted in modified spatial performance in the Morris water maze in male rats (Corrieri et al., 2007) and altered pain perception in female rats (Ceccarelli et al., 2009). Moreover, because of its widespread use and resistance to biodegradation, EE represents an environmental estrogen found in rivers of the United States and Europe as a contaminant from sewage treatment works (Kolpin et al., 2002; Pojana et al., 2004; Cargouët et al., 2004). Importantly, the exposure of fetal or neonatal rats to low doses of xenoestrogens (mimicking environmental concentrations) may affect their physiology and behavior in adulthood. In particular, low levels of EE affected the reproductive success of rats (Fusani et al., 2007), changing the sexual behavior of adult females (Della Seta et al., 2008). In our laboratory we also showed that prenatal exposure to low levels of BPA changes pain responses differently in male and female offspring during adulthood (Aloisi et al., 2002). In the present study we tested middle-aged (8-month-old) male and female rats that had been perinatally exposed to two different doses of EE through their mothers (in utero and during suckling) to better understand the long-term action of the treatment. We subjected the rats to the formalin test to evaluate the influence of low (mimicking the environmental concentration) or high (mimicking the contraceptive pill dose) perinatal xenoestrogen exposure on pain responses. Moreover, spontaneous behaviors were recorded four days later to study the long-lasting effect of the peripheral stimulation in the different groups. As in previous experiments in our laboratory, we used intact animals exposed to EE during pregnancy and lactation in order to cover all the critical rodent developmental period.

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oil, ensured that the treatment procedure was not stressful. Each female was then paired with a sexually mature male for 48 h in a Plexiglas cage (Techniplast, Italy, 60 × 37 × 20 cm) with a metal top and a wire net floor to allow the daily search for a vaginal plug. After detection of the vaginal plug (gestational day – GD – 0), the male was removed and the female was housed individually. On the day after mating and throughout the pregnancy (from GD 5 to GD 21) and during lactation (from PND 0 to PND 21), the females were treated daily with vehicle (OIL) or one of the two treatment concentrations. Substances Two doses of 17α-ethinylestradiol (EE, Sigma, Milan, Italy) dissolved in peanut oil (Sigma-Aldrich Milan, Italy) were used in this experiment. Treatments were carried out per os. The oral route of administration was selected because this is the main route of exposure in humans. The low 17α-ethinylestradiol dose (EEL: 4 ng/kg/day) matches EE concentrations found in contaminated surface waters (Nash et al., 2004; Parrott and Blunt, 2005), while the high dose (EEH, 400 ng/kg/day) is equivalent to that of most estrogenic or estro-progestinic contraceptive pills. The actual levels of EE that reached the fetus during gestation or were ingested by the pup during lactation were not determined in the present study. Peanut oil was used as vehicle (OIL). Experimental design Litters were reduced to 5 male and 5 female pups and left with the mother until weaning on PND 21, then juveniles of each litter were individually marked, separated and randomly housed in groups of four, according to sex, so that no cage contained siblings. Only one female and one male per litter were used in the present study. At 8 month of age, the offspring were subjected to the formalin test and (four days later) to the open field test. At the end of the open field test the animals were immediately anesthetized (sodium pentobarbital N70 mg/kg body weight) and the abdomen was opened to collect blood. All experimental procedures took place during the dark phase of the animals' circadian cycle, between 10:00 and 14:00 h, under dim light. Between tests the rats were kept in groups in their own cage.

Materials and methods

Vaginal cytology

Subjects

To decrease the variability among females, the formalin test was carried out while the females were in the estrus phase of their estrous cycle. Determination of the estrous cycle phase was carried out for each female every two days from the beginning to the end of the experiment. Male rats were similarly manipulated. The estrous cycle phase was determined by vaginal smears. Estrus was identified by the presence of a large number of cornified (or keratinized) cells, proestrus by the presence of clusters of round, nucleated epithelial cells, the first day of diestrus (diestrus 1) by a combination of leukocytes and cornified cells, and the second day of diestrus (diestrus 2) by the almost exclusive presence of leukocytes with small clumps of nucleated epithelial cells (Goldman et al., 2007).

Thirty-six female and 20 male Sprague–Dawley rats (Harlan Italy, Comerio, Italy) were used in the present study as reproductive subjects. Their offspring, born in the animal house of the Department of Physiology, University of Siena, were used to study the effect of EE exposure. In the present experiment, 58 male and 48 female offspring were used at about 8 months of age (body weight: 489 ± 4 g and 288 ± 4 g, respectively). Animals were housed in an air-conditioned room (temperature 21 ± 1 °C, relative humidity 60 ± 10%) with a 12-h light–dark cycle (lights on from 7.00 pm to 7.00 am). Water and food (Harlan Teklad diet) were available ad libitum. Attention was paid to the regulations of local authorities for handling laboratory animals, the European Communities Council Directive (86/609/EEC) and the Ethical Guidelines for investigation of experimental pain in conscious animals issued by the ad-hoc Committee of the International Association for the Study of Pain (Zimmermann, 1983). Mothers and their treatment Maternal treatment Prior to the experiment the females were trained for 5–6 days to suck peanut oil from a micropipette as previously described (Palanza et al., 2002). This training, together with the high palatability of the

Behavioral tests Formalin test The well-known formalin test (FT) is an experimental model of persistent pain (Aloisi et al., 1995; Capone and Aloisi, 2004). The subcutaneous injection of dilute formalin induces a series of quantifiable behavioral responses (licking, flexing and jerking of the injected hind paw) that allow the pain intensity and time course to be measured. The FT was carried out in Plexiglas cages (30 × 30 × 30 cm) and consisted in the subcutaneous injection of dilute formalin (FORM, 50 μl, 5%) into the right dorsal hind paw or a sham injection (SHAM) in which animals were merely pricked with the syringe needle without

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injection of any substance. The following groups (n = 7–10) were obtained: OIL FORM/OIL SHAM EEL FORM/EEL SHAM EEH FORM/EEH SHAM. Immediately after formalin or sham injection the rat was placed in the Plexiglas cage and its behavior was recorded for 60 min. During this period three temporal phases can be identified (the first phase from 0–5 min in which the animal shows a ‘burst’ of pain responses, especially licking, due to the chemical insult of nociceptors by formalin; an interphase from 5 to 20 min during which these responses decrease; the second phase, 20–60 min, during which there is a recovery of paininduced behavioral responses). The following spontaneous and formalin-induced behavioral responses were recorded: a) Formalin-induced responses: licking duration (time spent licking the injected foot), flexing duration (time spent with the leg held off the floor, flexed close to the body) and paw-jerk frequency (number of phasic flexions of the leg). b) Spontaneous behaviors: rearing frequency (number of times the animal stood on its fore limbs), grooming duration (time spent washing or scratching the face or body), activity duration (time spent sniffing and looking around the environment), sit alert duration (time spent motionless in an alert position), crouch duration (time spent motionless in a sleeping-like position). No pain-induced behaviors were manifested by the sham-injected animals and thus only spontaneous behaviors were considered for comparisons of SHAM and FORM groups. Open field test The open field (OF) test was carried out in the open field apparatus, a square transparent Plexiglas cage (50 × 50 cm) with 30 cm high walls situated on a platform 80 cm above the floor. The floor of the chamber is painted black and divided into 25 equal squares (16 outer and 9 inner) by intersecting white lines. The rat was kept in the open field for 5 min and the same spontaneous behaviors observed in the FT were recorded and analyzed; in addition, the numbers of crossings of the outer and inner white lines were recorded to evaluate possible changes in locomotor activity. A video-camera was used to record both tests. The recordings were later analyzed with Observer behavioral software (Noldus Information Technology, Amsterdam, the Netherlands) by a researcher blind to the treatment. At the end of the open field test, the animals were deeply anesthetized. Hormonal determinations The blood samples were kept in a refrigerator for about 3 h and then centrifuged to obtain serum that was immediately stored at − 20 °C until hormonal determinations. Testosterone and estradiol serum levels were determined by radioimmunoassay after extraction of serum with methylene chloride according to the method of Abraham (1975) with modifications. Briefly, tritiated estradiol and testosterone (NEN Life Science Products, Boston, MA, USA) were used as tracers together with the corresponding antibodies (Analytical Antibodies, Milan, Italy). Free and bound hormones were separated using charcoal-dextran. After centrifugation the radioactivity of the supernatant was determined with a beta counter. We employed a quality control procedure using known samples to check the efficiency of the method. The intra-assay coefficients of variation were 5%; to eliminate inter-assay variation, all samples were determined in the same assay. All samples were assayed in duplicate.

Data analysis Data are reported as mean ± SEM in the tables and figures. Analysis of variance (ANOVA) was applied to all recorded parameters with the factors Sex (2 levels: male, female), Treatment (3 levels: OIL, EEL, EEH) and Pain (2 levels: SHAM, FORM); the factor Time (repeated, 12 5-min periods) was used for the formalin-induced behavioral responses to evaluate the changes occurring during the formalin test. P b 0.05 was considered significant. Eta squared (η2) is reported for the ANOVA results; it was calculated by the formula η2: SSfactor/SStotal (SS = sum of squares). The Fisher Least Square Deviation (LSD) test was used for post-hoc analysis when necessary. All statistical analyses were carried out using the Stat Soft software, version 12.

Results Body weight To evaluate the effect of perinatal treatment on physical development, we weighed all animals belonging to the three treatment groups (OIL, EEL and EEH) at the beginning of the experimental phases when the animals were about 8 months old. The results show a significant effect of treatment in both sexes. While the weight in the EEL groups was similar to the OIL groups, the values were significantly lower in EEH groups (Table 1). The 2-way ANOVA revealed an effect of Sex (F(1,94) = 1218.01, p b 0.0001, η2 = 0.91), due to the lower body weight of females than males, and an effect of Treatment (F(2,94) = 7.09, p b 0.001, η2 = 0.01) due to lower body weight in the EEH groups than in the OIL groups independently of sex (even though the effect was more evident in females) (Table 1).

Estrous cycle To evaluate if prenatal exposure to EE induced changes in the estrous cycle and to select females to be tested while in estrus, the estrous cycle phase was determined every other day from the beginning until the end of the experiment. The results were: OIL groups (n = 14), 12 females were normally cycling, 2 were not cycling (persistent estrus phase); EEL groups (n = 20), 13 females were normally cycling and 7 not cycling; EEH groups (n = 14), all females were not cycling, indicating the loss of estral cyclicity.

Formalin test To better evaluate the effect of EE exposure in each sex, we first analyzed the duration/frequency of pain responses in males and females separately (repeated-ANOVA with the factors Treatment and Time). After that, two-way ANOVA with the factors Sex and Treatment was applied to the total values (licking, flexing and paw-jerk) recorded only during the second phase of the FT (from 20 to 60 min). The spontaneous behaviors were analyzed over the 60 min of the FT by repeated 3-way ANOVA with the factors Sex, Treatment and Pain.

Table 1 Body weight (g ± SEM) of male and female rats determined at the beginning of the experimental phases. Males were heavier than females independently of treatment (p b 0.0001). EEH exposure during gestation and lactation decreased body weight in both sexes (p b 0.001). Sex

Oil

EEL

EEH

Males Females

492.90 ± 8.06 291.57 ± 5.58

494.30 ± 5.95 301.65 ± 7.20

481.61 ± 7.81 264.00 ± 3.33

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Formalin test in males: effect of EE exposure on pain responses In males, perinatal EE exposure significantly affected pain behaviors, as both licking and flexing were higher in EE groups than OIL. For licking of the injected paw (Fig. 1A), ANOVA revealed a significant effect of Treatment (F(2,19) = 6.25, p = 0.0082, η2 = 0.05), due to the higher levels in EE groups than in OIL (p b 0.002 for EEH and p b 0.03 for EEL), and a significant Treatment × Time interaction (F(22,209) = 2.16, p b 0.003, η2 = 0.09) due to the higher levels in both EE groups than in OIL from 25 to 45 min of the FT. In particular, EEL showed higher levels of Licking than OIL during the second phase (at time 25, 30, both p b 0.02, and time 40, p b 0.001); similarly EEH showed higher levels of Licking than OIL during the second phase (at time 25, 45, both p b 0.01, and 35, 40, both p b 0.001); no differences were found during the first 20 min of the test (first and intermediate phase). For flexing (Fig. 1B), ANOVA revealed significance of the factor Treatment (F(2,19) = 9.48, p b 0.001, η2 = 0.12) and the Treatment × Time interaction (F(22,209) = 3.82, p = 0.0001, η2 = 0.10). This was due to the higher levels in EE groups than in OIL (both, p b 0.001) and to the presence of differences only during the second phase. Indeed higher levels of flexing in EEL were present at time 25, 50 and 55 (p b 0.03) and from time 30 to 45 (p b 0.001), whereas EEH showed higher levels from time 35 to 50 (p b 0.001) and at time 55 (p b 0.03). For paw-jerk (Fig. 1C), there were no statistically significant differences among groups. Formalin test in females: effect of EE exposure on pain responses In contrast to males, females were not significantly affected by treatment. Indeed ANOVA revealed no significant differences in any of the three pain responses (Fig. 1D–F, licking, flexing and paw-jerk) even though there was a clear sign of a decrease in licking and flexing duration in EEH with respect to OIL. Formalin test: pain responses among all male and female groups As evident from the previous analysis, pain behaviors showed significant differences among treatments in the second phase of the FT. Hence

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to evaluate possible sex-induced effects we considered the duration/frequency of each pain response only during this phase, i.e. from 20 to 60 min after formalin injection. For licking and flexing duration (Fig. 2A, B), there was a significant Sex × Treatment interaction (F (2,37) = 4.3, p b 0.02, η2 = 0.17 and F (2,37) = 3.32, p b 0.04, η2 = 0.12, respectively) due to the higher levels in the EE-exposed male groups with respect to OIL (licking: p b 0.01, flexing: p b 0.003) and the lower levels in EEH females than EEH males (licking: p b 0.005, flexing p b 0.01). Paw-jerk frequency (Fig. 2C) was higher in males than in females (Sex: F (1,37) = 16.89, p b 0.001, η2 = 0.29) independently of the treatment. It should be noted that also for this pain behavior the EEH groups were differently affected in the two sexes, with a strong tendency to higher levels than OIL in males and lower levels in females.

Formalin test: spontaneous behaviors in all groups (Table 2) ANOVA applied to explorative behavior revealed a significant Sex × Pain × Time interaction (F(11,781) = 1.79, p b 0.04, η2 = 0.009 for activity, F(11,781) = 2,20, p b 0.01, η2 = 0.008 for rearing); there were higher activity levels in females than males during the second phase of the FT, while in FORM groups the levels were higher in females and lower in males with respect to the SHAM ones. Immediately before sacrifice the animals were placed in the open field apparatus for 5 min to test the long-lasting effect of formalin injection in all groups (Table 3). EE exposure did not appear to induce longlasting changes, since only sex differences were found in explorative behaviors (inner and outer crossing, activity and rearing), with higher levels in females than in males (p b 0.002, 0.001, 0.002 and 0.001, and η2 = 0.13, 0.27, 0.09 and 0.20, respectively), and in sit alert duration which was higher in males than in females (p b 0.0002, η2 = 0.12) independently of treatment and pain. In contrast, grooming duration, higher in females than in males (Sex: F(1,94) = 4.85, p b 0.03, η2 = 0.04), was higher in EEH animals than in OIL independently of sex (Treatment: F(2,94) = 3.28, p b 0.04, η2 = 0.05).

Fig. 1. Formalin test: perinatal EE exposure increased pain responses only in males. Time course of pain-induced licking (A, D), flexing (B, E) and jerking (C, F) of the injected paw, in male (up) and female (bottom) rats perinatally exposed to OIL, EEL and EEH, during the 60 min of the formalin test. *p b 0.03 and **p b 0.001 EEL vs OIL; #p b 0.03 and ##p b 0.001 EEH vs OIL. Data are mean ± SEM.

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Fig. 2. Perinatal EE exposure significantly increased pain responses in males during the second phase of the formalin test. Total licking (A) and flexing (B) duration and pawjerk frequency (C) recorded during the second phase (20 and 60 min) of the FT in male and female rats perinatally exposed to OIL, EEL and EEH. *p b 0.01, **p b 0.003 vs OIL, same sex; #p b 0.01 vs females, same group. Data are mean ± SEM.

Gonadal hormones

Estradiol (Fig. 3A and D) As expected, estradiol serum levels were higher in females than males and, only in females, lower in formalin-treated groups exposed to EE. In particular ANOVA revealed significant effects of Sex (F(1,86) = 60.7, p b 0.001, η2 = 0.35) and Pain (F(1,86) = 6.06, p b 0.015, η2 = 0.035) and a Sex × Treatment × Pain interaction (F(2,86) = 3.59, p b 0.032, η2 = 0.041). This was due to the higher

Table 2 Total duration/frequency of spontaneous behaviors recorded during the formalin test in males and females exposed to OIL, EEL or EEH during the perinatal period. See results section for details. Data are mean ± SEM. Behavior

Males

Females

Sham

ACTIVITY (s) SIT ALLERT (s) GROOMING (s) REARING (n) CROUCH (s)

Formalin

Sham

Formalin

OIL

EEL

EEH

Oil

EEL

EEH

Oil

EEL

EEH

Oil

EEL

EEH

2114.2 ± 93.9 868.2 ± 190.2 231.4 ± 54.2 78.2 ± 7.8 287.3 ± 110.2

1885.7 ± 147.3 692.0 ± 55.5 256.9 ± 22.5 69.6 ± 10.2 688.5 ± 161.6

2026.2 ± 114.7 719.0 ± 132.4 387.1 ± 101.9 78.7 ± 7.9 449.5 ± 175.7

1964.8 ± 131.2 736.6 ± 136.9 281.4 ± 29.7 53.0 ± 5.7 392.9 ± 174.3

1831.1 ± 181.0 470.2 ± 143.6 265.3 ± 34.1 47.1 ± 4.4 628.3 ± 142.0

1634.4 ± 90.5 762.7 ± 97.5 229.1 ± 23.9 55.4 ± 6.5 571.6 ± 116.9

2325.6 ± 187.2 952.9 ± 165.9 396.7 ± 76.8 107.2 ± 10.1 12.4 ± 9.9

2145.3 ± 126.7 927.4 ± 90.6 474.8 ± 46.4 95.0 ± 11.9 48.9 ± 47.9

2350.7 ± 212.5 709.9 ± 194.4 522.6 ± 57.2 95.2 ± 18.4 0.0

2285.7 ± 113.8 561.7 ± 91.3 367.7 ± 32.3 102.9 ± 12.6 28.3 ± 16.7

2370.4 ± 198.4 635.0 ± 144.7 261.7 ± 48.0 81.6 ± 21.5 65.8 ± 32.2

2449.3 ± 184.1 342.5 ± 64.7 444.6 ± 68.5 97.0 ± 21.6 92.1 ± 73.5

49.3 ± 3.8 133.4 ± 7.2 256.2 ± 14.9 9 ± 2.7 34.8 ± 15.9 40.4 ± 3.5 44.1 ± 3.7 133.7 ± 7.9 273.7 ± 5.3 13.5 ± 4.7 12.8 ± 2.8 31.5 ± 1.8 47.9 ± 4.7 142.1 ± 7.5 276.8 ± 5 8.2 ± 2.7 14.9 ± 4.6 30.5 ± 2.2 51.1 ± 5.8 105.4 ± 12.7 261.8 ± 27.7 17.7 ± 17.4 20.5 ± 11 33.1 ± 3.6 31.9 ± 5.5 95.9 ± 8.7 260.2 ± 13.1 17.4 ± 6.5 22.4 ± 11.1 26.9 ± 3 35 ± 4.1 95.7 ± 8.4 242.5 ± 19.6 39.7 ± 22.1 18.1 ± 5.8 26 ± 3.5 36.8 ± 6.7 94.4 ± 7.2 226 ± 22.4 63.9 ± 24.52 10.7 ± 3.5 26.6 ± 2.2 34 ± 4.7 92.5 ± 5 242.1 ± 14.5 48 ± 16.1 10 ± 4.2 24.9 ± 2.1 39.6 ± 5.2 94.7 ± 6.1 216.8 ± 27.7 82.4 ± 28 0.77 ± 0.6 26.8 ± 3.3 33.2 ± 5.4 106.8 ± 10.4 209.9 ± 27.9 65.4 ± 27.2 4.9 ± 4.1 22.4 ± 3 Internal crossing (n) External crossing (n) Activity (s) Sit alert (s) Grooming (s) Rearing (n)

EEH EEL EEH EEL

48.7 ± 8.3 139 ± 27.7 265.9 ± 10.1 5.6 ± 2.5 28.5 ± 11.9 39.2 ± 5.7

43.8 ± 6.2 132.6 ± 8.6 278.5 ± 8.87 13.7 ± 7.6 7.8 ± 2.7 35 ± 3.4

EEH EEL Formalin

Oil EEH oil

EEL Sham

oil

Sham

Oil

Females

Formalin Males Behavior

Table 3 Spontaneous behaviors recorded during the Open Field test (5 min) in males and females exposed to OIL, EEL or EEH during the perinatal period. See results section for details. Data are mean ± SEM.

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estradiol levels in females than in males of each group (p b 0.01 for all), except the EEH-FORM groups; moreover FORM EEL and EEH females had lower estradiol levels than FORM OIL (p b 0.001) and the corresponding SHAM groups (p b 0.01) (Fig. 3A and D). In SHAM females, the EE groups showed a tendency to higher levels than OIL. Testosterone (Fig. 3B and E) Testosterone serum levels did not show significant changes in EEexposed animals. Indeed ANOVA revealed a significant effect of Sex (F(1,93) = 61.92, p b 0.0001, η2 = 0.38) due to the higher levels in males than in females independently of treatment or pain (Fig. 3B and E). Estradiol/testosterone ratio (E/T, Fig. 3C and F) The E/T ratio differed significantly between the sexes and was affected by treatment. ANOVA revealed significant effects of Sex (F (1,85) = 99.07, p b 0.0001, η2 = 0.46), Treatment (F (2,85) = 5.00, p b 0.008, η2 = 0.046) and Pain (F (1,85) = 5.30, p b 0.02, η2 = 0.025), as well as Sex × Treatment (F (2,85) = 5.36, p b 0.006, η2 = 0.05) and Sex × Pain interactions (F (1,85) = 4.34, p b 0.04, η2 = 0.02). This was due to higher values in females than in males independently of treatment, to EEL and EEH females having significantly lower values than OIL (p b 0.0001 and p b 0.004, respectively) and to lower values in FORM than in SHAM females (p b 0.02). Discussion The main finding of this study is the demonstration of the ability of 17α-ethinylestradiol (EE) administered to pregnant and lactating female Sprague Dawley rats to induce long-term effects in their offspring during adulthood. However, at 8 months of age, the male and female offspring were affected differently by the treatment, especially with regard to the nociceptive system (as shown by the formalin test). In male offspring there was an increase in pain-induced licking and flexing (changes in the female direction) while in females these responses showed no differences or a tendency to decrease (changes in the male direction). Studies on rodents and humans have clearly shown that estrogens can modulate pain, with the changes varying depending on the sex of the animals studied, the estrogen concentration and the pain model used (Craft, 2007; Aloisi et al., 2010; Smith et al., 2006). For instance, estrogen can be pro-nociceptive at low, physiological levels but antinociceptive at higher levels. The pro-nociceptive role of estrogens is supported by previous experiments in which we demonstrated that: a) for most of the formalin concentrations tested, females show higher pain responses than males (Aloisi et al., 1995), b) there is an increase in licking behavior in male rats after estrogen priming in the CNS (Aloisi and Ceccarelli, 2000), and c) chronic pain can develop in transsexual men taking estrogens (Aloisi et al., 2007). As for the antinociceptive effect played by estrogen, it was observed in females exposed to high EE concentrations, which probably mimic the higher estrogenic conditions obtained at experimental levels in rats (Aloisi et al., 2010) or in humans (Smith et al., 2006) or during pregnancy (Liu and Gintzler, 1999). Prenatal (Cao et al., 2013), perinatal (Ceccarelli, personal communication) and pubertal (Ceccarelli et al., 2007) exposure to estrogenic compounds has been found to increase ER-α levels in many brain regions in male and female rats. Thus our first hypothesis concerning the possible systems modulated by EE exposure involves ER-sensitive pathways in the CNS. As clearly shown by the changes in the formalin-induced pain responses, EE-exposed male rats did not show higher pain responses from the beginning of stimulation (the first phase, induced by peripheral nociceptor activation) but in the second phase of the formalin test when pain responses are due not only to persistence of the stimulus but also to central modulation. For this reason, supraspinal centers, and the septo-hippocampal pathway in particular, must be involved. This pathway mediates arousal and attention states

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Fig. 3. Gonadal hormones. Histograms of 17-β estradiol (A, D), testosterone (B, E) and E/T (C, F) serum levels in male and female rats perinatally exposed to OIL, EEL and EEH. #p b 0.001 vs OIL FORM; §p b 0.01, vs Sham, same treatment. Data are mean ± SEM.

(Sutherland and McNaughton, 2000) and estrogens and formalin pain are known to modulate it by increasing ACh release, spine density and connections (Ceccarelli et al., 1999; Frick, 2015; Sellers et al., 2015); all events that can contribute to the higher formalin-induced pain behavior present in female than in male rats (Aloisi et al., 1996). Thus the increase of licking and flexing found in males during the second phase of the formalin test could be due to ER-induced greater attention to the nociceptive stimulus, as usually present in females. Further confirmation of this supraspinal modulation of pain in males comes from the fact that paw-jerk, used as a marker of spinally mediated nociceptive inputs, is always higher in males than in females and is not modulated by EE; this is in agreement with previous observations in males treated with estradiol (Ceccarelli et al., 2004). EE exposure in females had different effects. Not only was there no sign of an increase but the group treated with the higher EE concentration (EEH) showed a non-significant decrease with respect to the other two groups. This tendency was also present for paw-jerk frequency. On the whole these data strongly suggest that the EE-induced changes in females can be compared to the estrogen-induced analgesia present when females have high levels of estrogen in the blood (Aloisi et al., 2010; Liu and Gintzler, 1999; Smith et al., 2006; Mannino et al., 2007). Indeed it has been shown that anti-nociception varies during the different phases of the estrous cycle in female rats. Moreover opioid/estrogen interactions in the nociceptive system, thought to be mediated at spinal levels (Gintzler et al., 2008), represent an important aspect that must be considered (Kepler et al., 1989; Lee and Ho, 2013). Finally, since the higher pain responses found in males are similar to those found after prenatal, but not postnatal, exposure to BPA (Aloisi et al., 2002), it can be hypothesized that the most important effects on the pain system occur more during the prenatal period than the lactating phase. The disruption of the normal estrous cycle in all offspring of mothers administered the clinically used dose of EE (400 ng/kg/day) confirms previous reports of suppressive effects of EE exposure from gestation to puberty on female sexual activity and the estrous cycle (Fusani et al., 2007; Della Seta et al., 2008). The same effects on the female estrous cycle and reproductive organs were reported by Sawaki but only with an EE dose 100-fold higher than the one we used (Sawaki et al., 2003). Moreover a recent paper showed an increase in the percentage

of animals found in the estrus phase at PND 180 after a single administration (at PND 0) of EE at two very high doses (20 and 2000 μg/kg) (Nozawa et al., 2014). The estradiol serum levels measured in all groups are further evidence of possible negative effects of EE exposure on CNS functions. Although male and female estradiol values at 8 months of age were slightly lower than those found in females at PND 90, i.e. in reproductive age (Della Seta et al., 2008), the estradiol levels were significantly changed by EE exposure in females and not modified in males, as also found by Howdeshell et al. (2008). Regarding the changes observed in females, while EE exposure tended to increase estrogen levels with respect to OIL in the control pain-free groups, in EE-exposed females subjected to the formalin test the estradiol levels were drastically decreased. These changes suggest interference not only with reproductive aspects but also with nociception. Indeed it is well known that supraspinal brain areas involved in pain modulation, i.e. the arcuate nucleus of the hypothalamus, are also involved in HPG modulation (see Aloisi and Ceccarelli, 2000). No effect of EE exposure was found on estrogen levels in sham-injected animals tested in the estrus phase of their cycle or in the formalin-treated OIL groups. Instead, formalin pain per se was able to greatly decrease estrogen levels, probably through the interaction between estrogens and POMC neurons; while estrogens are able to increase β-endorphin release, which plays an important role in inhibiting GnRH neurons (Lagrange et al., 1995), the concomitant effect on POMC neurons of the painful stimulation would have amplified the inhibition of the HPG axis. This is interesting since it appears that EE-exposed females are more vulnerable to stress and/or inflammation, i.e. their HPG axis not only is blocked and not cycling but also seems more responsive to insult. A significant decrease in body weight in adult male rats (4 months old) perinatally exposed to a high dose of EE (50 μg/kg/day) was reported by Howdeshell and by Sawaki in two different rat strains, Lewis and Sprague–Dawley respectively (Howdeshell et al., 2008; Sawaki et al., 2003). More recently, Ferguson et al. (2014), using a lower concentration (10 μg/kg), found a significant weight increase in female Sprague–Dawley rats at PND 100. In the present experiment on older rats (8 months old), there was a weight decrease in both sexes, albeit more evident in females, and it was found at a lower dose (400 ng/kg/day) than those used by the above-mentioned authors,

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confirming data reported by Fusani in 7-month-old Sprague–Dawley females treated with EE (400 ng/kg/day) from gestation to puberty (Fusani et al., 2007). Interestingly these data can be explained by the positive effects of estrogens on energy balance and regulation of feeding exerted at hypothalamic levels through ERα(see Xu et al., 2011). Behavior is the final point of confluence of complex integrated systems that can be influenced by subtle environmental alterations. The study of behavior, supplemented by the analysis of neuroendocrine parameters, can provide indications about the effect of xenoestrogens in the developmental phase in which the behavioral circuits are organized (Della Seta et al., 2008). With the present study we show important sex differences in these effects. Acknowledgments This work was supported by MIUR (20088NJR3B_003) (Italian Ministry for Research and University) to A.M.A.. 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