Maternal exposure to fluoxetine during gestation and lactation induces long lasting changes in the DNA methylation profile of offspring’s brain and affects the social interaction of rat

A.S. Silvaa, L.V. Toffolib, V.B. Estradaa, L.F. Veríssimoa, J. Francis-Oliveiraa, E.G. Moreiraa

Abstract

Fluoxetine (FLX) is an antidepressant from the selective serotonin reuptake inhibitor class that has largely been used for the treatment of depression in pregnancy. However, increasing evidences have indicated the potential of early maternal exposure to FLX to induce molecular and neuro functional effects on the offspring. In the present study we evaluated possible long lasting impacts of the maternal exposure to FLX during gestation and lactation. Female Wistar rats were gavaged with 5 mg/kg of FLX during the period that comprehends the first day of pregnancy (PD0) and the last day of lactation (LD21) (Group FLX). Control group (CTL) received a proportional volume of water. On the postnatal day 75 (PND75), male rats were euthanized and hippocampus, cortex, hypothalamus, and periaqueductal gray area (PAG) were removed. Global DNA methylation was quantified using a high-throughput ELISA-based method. In order to address neuro functional changes animals (PND75) were evaluated in the elevated plus maze and social interaction tests as well as submitted to repeated restraint stress. An increase in the global DNA methylation profile of hippocampus (p = 0.0399) was associated with the early exposure to FLX, whereas no significant change was observed in the hypothalamus (p = 0.6556), cortex (p =0.9402) or PAG (p =0.3822). Furthermore, early exposure to FLX was also associated with a reduction in the social interaction time (p = 0.0084) and to a decreased in the plasma corticosterone level when animals were submitted to the restraint stress (p < 0.0001). No significant change in the elevated plus maze test was associated with the early exposure to FLX. In summary, our data demonstrate that maternal exposure to FLX during gestation and lactation results in a long lasting impact on the DNA methylation of hippocampus, and affects the social behavior and the corticosterone response to stress.

Keywords:
Fluoxetine
DNA methylation
Epigenetics
Gestation
Lactation

1. Introduction

Fluoxetine (FLX) is an antidepressant of the class of serotonin selective reuptake inhibitors (SSRIs), widely prescribed worldwide for the treatment of affective and anxiety disorders, and due to its relative selectivity of action, efficacy and reduced side effects it is one of the drugs of choice during pregnancy (Nonacs and Cohen, 2003).
Although it is currently known that FLX crosses the placenta (Pohland et al., 1989) and is excreted in breast milk (Hendrick et al., 2001), most women cannot interrupt the treatment during pregnancy and lactation. Thus, fetuses and newborns end up being exposed to this drug at critical stages of neurodevelopment, in which serotonin (5-HT, 5-hydroxytryptamine) exerts a trophic influence for migration and synaptogenesis of monoaminergic neurons (Whitaker-Azmitia et al., 1996).
Studies in animals have suggested that perinatal SSRI administration can induce anxiety and depressive-like behavior in rodents (Lisboa et al., 2007; Popa et al., 2008) suggesting that exposure to FLX during neurodevelopment may have a lasting impact on brain function. The perception that events that occur during development, including exposure to drugs, may be involved with the onset of late diseases led to the proposition of the “Developmental Origin of Health and Disease (DOHaD) theory. This paradigm is based on the influence of environment on embryo-fetal and postnatal period and the morphological development of the organs and the emergence of diseases in adulthood (Gluckman et al., 2008). The biological mechanisms behind the relationship between events occurring in development and its effects on long-term health are poorly understood. Yet a growing body of evidence points to some candidate mechanisms, including changes in gene expression via epigenetic mechanisms (Martin-Gronert and Ozanne, 2012; Junien et al., 2016).
Our group demonstrated the vulnerability of the DNA methylation programming in weaned rats maternally exposed to FLX during gestation and lactation (Toffoli et al., 2014). Nevertheless information about the persistence of the abnormal epigenetic patterns and the implications to the brain function later in life is still lacking.
In this context, the present study aimed to evaluate the effects of the exposure to FLX during gestation and lactation on the DNA methylation of brain regions and on behavioral parameters in adult animals.

2. Materials and methods

Female Wistar rats were gavaged with 5 mg/kg/day of FLX during the first day of pregnancy (PD0) to the last day of lactation (post-natal day 21- PND21) (FLX group). Control group (CTL) received a proportional volume of water during the same period. Weights of pregnant females were obtained every day. Each offspring was reduced to 8 animals on the PND4 to avoid bias related to the access to FLX via lactation.
Animals were kept in a controlled environment, with temperature at 21 ± 2 °C, 12 h dark/light cycle (lights on at 06:00 am) and free access to food and tap water. Procedures were performed with the approval of the Ethics Committee for Animal Experimentation of the State University of Londrina (nº17142/11).
On the PND 75, male rats from FLX and CTL groups obtained from different litters (litter was considered the experimental unit) were submitted to the behavioral tests (elevated plus maze (Pellow and File, 1986) or social interaction (Moreira et al., 2001)) or were euthanized by decapitation for samples collection (i.e., hippocampus, hypothalamus, cortex, PAG and blood). Samples were immediately frozen at −80 °C. DNA extractions from encephalic samples were performed by standard salting out protocol. The Paxinos and Watson Atlas (Paxinos and Watson, 2009) was considered as neuroanatomic reference. All protocols were conducted between 08.00 and 12.00 h in the light phase and each animal was submitted to only one behavioral test.
Global DNA methylation profile was evaluated by dosage of the methyl groups (CH3) using the Imprint Methylated DNA Quantification Kit (Sigma-Aldrich®), as previously described (Toffoli et al., 2014).
The Elevated Plus-Maze is a test for assessment of anxiety that is based on the time spent by the animal in an environment initially aversive (open arms) and a protective environment (closed arms). The elevated plus-maze apparatus was made of wood and consisted on two opposed open arms, 50 x 10 cm (length × width), crossed with two opposed closed arms of the same size, but enclosed by walls (40 cm high), except for the entrance. The apparatus was elevated 50 cm above the floor. To prevent falls, the open arms were surrounded by a Plexiglas rim 1 cm high. In the experiment room, illumination was provided by a 60 W incandescent lamp in the ceiling. For the test, each animal was placed in the center of the maze, facing one of the open arms, and the number of entries into, and the time spent in the open and closed arms were registered for 5 min (Pelosi et al., 2009).
The social interaction test was carried in a circular wood arena (72 cm of diameter) surrounded by a wall. Adult rats exposed to the same regimen were housed in pairs for 7 days prior to the test. The test consisted of familiarizing each pair (cage mates) of rats with the arena for a period of 8 min on 2 consecutive days. On the third day, each rat was randomly assigned to a no familiar partner (but from the same exposure regimen) according to weight. These animals were placed in the arena to observe social interaction behavior for 5 min. Social interaction time (in seconds) per pair of rats was measured as the time spent sniffing the partner, climbing over and crawling under the partner, mutual grooming, genital investigation, and following and walking around the partner (Moreira et al., 2001). Aggressive behavior was not considered to be a social interaction behavior.
Animals were subjected to the repeated restraint stress protocol for 5 consecutive days, being placed in a metal cylinder 6.5 cm in diameter and 15 cm long with holes that allow ventilation, where they remained fixed and were kept enclosed for 60 min (Kashimoto et al., 2016). Blood samples were collected in heparinized (Heptar, 5.000 UI/mL, Eurofarma, Brazil) tubes and centrifuged at 2300 rpm for 20 min and the plasma frozen for subsequent plasma corticosterone level analysis by radioimmunoassay (Saia et al., 2011).
The analysis of the normality of the data was performed using the Shapiro-Wilk test. The non-parametric test of Mann-Whitney was used to compare data from global DNA methylation, social interaction, elevated plus maze and plasma corticosterone level. We used the software GraphPad Prism 5 for statistical analysis and considered p < 0.05.

3. Results

No effects on the weight of pregnant rats, live births and weight or physical development of offspring was associated with FLX treatment (data not shown) indicating lack of reproductive toxicity of the treatment.
A statistically significant increase in the global DNA methylation profile was observed in the hippocampus (p = 0.0399) from FLX-exposed animals in comparison to CTL. No significant difference was observed when compared the methylation profiles of hypothalamus (p = 0.656), cortex (p = 0.9402) or PAG (0.3822) (Fig. 1).
No significant difference was observed when compared the behavioral response of animals from FLX and CTL groups in the elevated plus maze test (Fig. 2). However, a statistically significant decrease in the social interaction time was observed in animals from FLX (25 ± 13/36, n =9) compared to CTL group (49 ± 35/63, n =8; p =0.0002] indicating the association between maternal exposure to FLX and an anxiogenic-like effect on adult rat offspring (Fig. 3).
Repeated restraint stress increased the plasma corticosterone level of CTR (no stressed: 2.9 ± 0.45/16.1, n =12, stressed: 20.3 ± 14.9/11.6, n =12; p < 000.1) and FLX group (no stressed: 0.88 ± 0.42/ 7.4, n =10, stressed: 16.2 ± 7/19.8, n =11; p < 0.0001). A significant decrease of the plasma corticosterone level (p < 0.0001) was observed when compared the adaptive responses of animals from FLX and CTL groups to repeated restraint stress (Fig. 4).

4. Discussion

Recently, we showed that the exposure to FLX during pregnancy and lactation affects the DNA methylation profile of specific brain areas such as the hippocampus and cortex in weaned rats (PND 22) (Toffoli et al., 2014). However, the information about the impact of the early FLX-induced changes on the encephalic DNA methylation on the mature brain was still lacking.
In the present study, we shown that the change in DNA methylation induced by the exposure to FLX during pregnancy and lactation persists in the hippocampus of offspring until adulthood (PND 75). Thus confirming that hippocampus, a brain region that plays a key role in learning, memory and stress responses, might be particularly sensitive to environmental changes (Collins et al., 2009) and retains the early induced epigenetic patterns over lifetime.
The relationship between early exposure to FLX and behavioral changes in adulthood is currently on the focus of debates (FrancisOliveira et al., 2013). Whereas some reports propose that exposure to FLX during gestation and lactation associates to changes in the anxiety and/or depression-like in pubescents and adult rats, contradictory evidences have questioned this hypothesis (Francis-Oliveira et al., 2013; Karpova et al., 2009). Furthermore, the differential experimental
Whiskers represent the min and max values. conditions by which the current data were obtained, such as drug dosage, route of administration, period of exposure and age at treatment, cannot be excluded from the uncontrolled factors that might be involved with discrepancies. Future studies are certainly needed for a complete understanding on this field.
The pathogenesis of anxiety disorders is complex and involves interaction of biological, environmental and psychosocial mechanisms (Domschke, 2014). In a recent study, Non et al (Non et al., 2014) used a high throughput array-based approach to verify epigenome-wide DNA methylation changes in umbilical cord blood cells of neonates exposed to non-medicated maternal depression or anxiety, or selective serotonin reuptake inhibitors (SSRIs) during pregnancy. They identified 42 CpG sites with significantly different DNA methylation levels in neonates exposed to non-medicated depression or anxiety relative to controls, somehow corroborating the notion of an association between DNA methylation changes and depression or anxiety (Non et al., 2014). Furthermore, the author verified that CpG site methylation was not significantly different in neonates exposed to SSRIs relative to the controls revealing the potential of SSRI in modulating the epigenetic profiles in offspring (Non et al., 2014).
Corroborating, a recent study using animal model demonstrated that FLX attenuates changes in DNA methylation levels in the hippocampus consequent to stress, thus demonstrating the participation of epigenetic mechanisms in the molecular pathway by which FLX exerts
Within this context, the data from the present study corroborate the notion of the potential of SSRI to modulate epigenetic mechanisms and provide additional information to the current understanding by indicating that early exposure to FLX induces lasting epigenetic changes that might be associated with the pathogenesis of late onset anxiety. However, future studies on this field are certainly needed for a complete elucidation of this proposition.
Stress has been considered an important model for the analysis of the participation of DNA methylation on the control of behavioral changes. Hippocampal changes of the DNA methylation that are evoked by stress might be attenuated by treatment with FLX and DNA methyltransferase inhibitors (DNMTi) supporting the role of epigenetic mechanisms on behavioral response (Sales and Joca, 2016). Furthermore, recent studies have shown that both prenatal maternal stress and cortisol might influence on infant stress regulation in the offspring (Abe et al., 2007; Kapoor et al., 2008).
In humans, stress and maternal depression have been associated with stress neonatal amended regulation, with the methylation pattern modulation of the human glucocorticoid receptor (GR) gene (NR3C1) in newborns, and with elevated baseline levels of salivary or urinary cortisol in children (Davis et al., 2011; Oberlander et al., 2008)
Besides, prenatal stress enhances fear-like behavioral profile and dysregulation of the HPA axis activity in rats (Green et al., 2011). However, administration of FLX during the early postnatal period can normalize plasma corticosterone response to stress in prenatally stressed mouse offspring (Ishiwata et al., 2005).
In the present work, maternal FLX exposure induced a decrease in the corticosterone level of stressed adult pup confirming the modulatory effect of FLX on HPA axis activity.

5. Conclusion

Taken together, our data suggest that the mechanism of action of FLX may involve epigenetic components in specific areas of the brain like the hippocampus, which play a role on the modulation of anxietyrelated behaviors; furthermore, FLX exposure during development induces a long lasting effect on the HPA system including changes in the levels of corticosterone in the blood.

References

Abe, H., Hidaka, N., Kawagoe, C., Odagiri, K., Watanabe, Y., Ikeda, T., Ishizuka, Y., HashiguchI, H., Takeda, R., Nishimori, T., Ishida, Y., 2007. Prenatal psychological stress causes higher emotionality, depression-like behavior, and elevated activity in the hypothalamopituitary-adrenal axis. Neurosci. Res. 59 (2), 145–151.
Collins, A., Hill, L.E., Chandramohan, Y., Whitcomb, D., Droste, S.K., Reul, J.M., 2009. Exercise improves cognitive responses to psychological stress through enhancement of epigenetic mechanisms and gene expression in the dentate gyrus. PLoS One 4 (1), e4330.
Davis, E.P., Glynn, L.M., Waffarn, F., Sandman, C.A., 2011. Prenatal maternal stress programs infant stress regulation. J. Child Psychol. Psychiatry 52, 119–129.
Domschke, K., 2014. Predictive factors of anxiety disorders. Nervenarzt 85, 1263–1268.
Francis-Oliveira, J., Ponte, B., Barbosa, A.P., Veríssimo, L.F., Gomes, M.V., Pelosi, G.G., Britto, L.R., Moreira, E.G., 2013. Fluoxetine exposure during pregnancy and lactation: effects on acute stress response and behavior in the novelty-suppressed feeding are age and gender-dependent in rats. Behav. Brain Res. 252, 195–203.
Gluckman, P.D., Hanson, M.A., Cooper, C., Thornburg, K.L., 2008. Effect of in utero and earlylife conditions on adult health and disease. New Engl. J. Med. Surg. Collat. Branches Sci. 359, 61–73.
Green, M.K., Rani, C.S., Joshi, A., Soto-Piña, A.E., Martinez, P.A., Frazer, A., Strong, R., Morilak, D.A., 2011. Prenatal stress induces long term stress vulnerability, compromising stress response systems in the brain and impairing extinction of conditioned fear after adult stress. Neuroscience 192, 438–451.
Hendrick, V., Stowe, Z.N., Altshuler, L.L., Mintz, J., Hwang, S., Hostetter, A., Suri, R., Leight, K., Fukuchi, A., 2001. Fluoxetine and norfluoxetine concentrations in nursing infants and breast milk. Biol. Psychiatry 50, 775–782.
Ishiwata, H., Shiga, T., Okado, N., 2005. Selective serotonin reuptake inhibitor treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction. Neuroscience 133, 893–901.
Junien, C., Panchenko, P., Pirola, L., Amarger, V., Kaeffer, B., Parnet, P., Torrisani, J., Jimenez, F.B., Jammes, H., Gabory, A., 2016. The new paradigm of the developmental origin of health and diseases (DOHaD) – epigenetics and environment: evidence and missing links. Med. Sci. 32, 27–34.
Kapoor, A., Petropoulos, S., Matthews, S.G., 2008. Fetal programming of hypothalamic–pituitary–adrenal (HPA) axis function and behavior by synthetic glucocorticoids. Brain Res. Rev. 57 586–59.
Karpova, N.N., Lindholm, J., Pruunsild, P., Timmusk, T., Castrén, E., 2009. Long-lasting behavioural and molecular alterations induced by early postnatal fluoxetine exposure are restored by chronic fluoxetine treatment in adult mice. Eur. Neuropsychopharmacol. 19, 97–108.
Kashimoto, R.K., Toffoli, L.V., Manfred, M.H.F., Volpini, V.L., Martins-Pinge, M.C., Pelosi, G.G., Gomes, M.V., 2016. Physical exercise affects the epigenetic programming of rat brain and modulates the adaptive response evoked by repeated restraint stress. Behav. Brain Res. 296, 286–289.
Lisboa, S.F.S., Oliveira, P.E., Costa, L.C., Venâncio, E.J., Moreira, E.G., 2007. Behavioral evaluation of male and female mice pups exposed to fluoxetine during pregnancy and lactation. Pharmacology 80, 49–56.
Martin-Gronert, M.S., Ozanne, S.E., 2012. Mechanisms underlying the developmental origins of disease. Rev. Endocr. Metab. Disord. 13, 85–92.
Moreira, E.G., Vassilieff, I., Vassilieff, V.S., 2001. Developmental lead exposure: behavioral alterations in the short and long term. Neurotoxicol. Teratol. 23, 489–495.
Non, A.L., Binder, A.M., Kubzansky, L.D., Michels, K.B., 2014. Genome-wide DNA methylation in neonates exposed to maternal depression, anxiety, or SSRI medication during pregnancy. Epigenetics 9, 964–972.
Nonacs, R., Cohen, L.S., 2003. Assessment and treatment of depression during pregnancy: an update. Psychiatr. Clin. North Am. 26, 547–562.
Oberlander, T.F., Weinberg, J., Papsdorf, M., Grunau, R., Misri, S., Devlin, A.M., 2008. Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses. Epigenetics 3, 97–106.
Paxinos, G., Watson, C., 2009. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego.
Pellow, S., File, S.E., 1986. Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacol. Biochem.Behav. 24, 525–529.
Pelosi, G.G., Resstel, L.B.M., Soares, V.P., Zangrossi, H., Guimarães, F.S., Corrêa, F.M.A., 2009. Anxiolytic-like effect of noradrenaline microinjection into the dorsal periaqueductal gray of rats. Behav. Pharmacol. 20 (3), 252–259.
Pohland, R.C., Byrd, T.K., Hamilton, M., Koons, J.R., 1989. Placental transfer and fetal distribution of fluoxetine in the rat. Toxicol. Appl. Pharmacol. 98, 198–205.
Popa, D., Léna, C., Alexandre, C., Adrien, J., 2008. Lasting syndrome of depression produced by reduction in serotonin uptake during postnatal development: evidence from sleep, stress, and behavior. J. Neurosci. 28, 3546–3554.
Saia, R.S., Oliveira-Pelegrin, G.R., da Silva, M.E.N.B., Aguila, F.A., Antunes-Rodrigues, J., et al., 2011. Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats. Regul. Pept. 169, 21–30.
Sales, A.J., Joca, S.R., 2016. Effects of DNA methylation inhibitors and conventional antidepressants on mice behaviour and brain DNA methylation levels. Acta Neuropsychiatr. 28, 11–22.
Toffoli, L.V., Rodrigues JR, G.M., Oliveira, J.F., Silva, A.S., Moreira, E.G., Pelosi, G.G., Gomes, M.V., 2014. Maternal exposure to fluoxetine during gestation and lactation affects the DNA methylation programming of rat´s offspring: modulation by folic acid supplementation. Behav. Brain Res. 265, 142–147.
Whitaker-Azmitia, P.M., Druse, M., Walker, P., Lauder, J.M., 1996. Serotonin as a developmental signal. Behav. Brain Res. 73, 19–29.