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Sep 8, 2015 - Citalopram and sertraline exposure compromises embryonic bone development. D Fraher1,2,6, JM Hodge2,3,6, FM Collier2, JS McMillan3, RL ...
Molecular Psychiatry (2016) 21, 656–664 © 2016 Macmillan Publishers Limited All rights reserved 1359-4184/16 www.nature.com/mp

ORIGINAL ARTICLE

Citalopram and sertraline exposure compromises embryonic bone development D Fraher1,2,6, JM Hodge2,3,6, FM Collier2, JS McMillan3, RL Kennedy3, M Ellis1,2, GC Nicholson3, K Walder1,2, S Dodd2, M Berk2,4, JA Pasco2,5, LJ Williams2,7 and Y Gibert1,2,7 Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed treatments for depression and, as a class of drugs, are among the most used medications in the world. Concern regarding possible effects of SSRI treatment on fetal development has arisen recently as studies have suggested a link between maternal SSRI use and an increase in birth defects such as persistent pulmonary hypertension, seizures and craniosynostosis. Furthermore, SSRI exposure in adults is associated with decreased bone mineral density and increased fracture risk, and serotonin receptors are expressed in human osteoblasts and osteoclasts. To determine possible effects of SSRI exposure on developing bone, we treated both zebrafish, during embryonic development, and human mesenchymal stem cells (MSCs), during differentiation into osteoblasts, with the two most prescribed SSRIs, citalopram and sertraline. SSRI treatment in zebrafish decreased bone mineralization, visualized by alizarin red staining and decreased the expression of mature osteoblast-specific markers during embryogenesis. Furthermore, we showed that this inhibition was not associated with increased apoptosis. In differentiating human MSCs, we observed a decrease in osteoblast activity that was associated with a decrease in expression of the osteoblast-specific genes Runx2, Sparc and Spp1, measured with quantitative realtime PCR (qRT-PCR). Similar to the developing zebrafish, no increase in expression of the apoptotic marker Caspase 3 was observed. Therefore, we propose that SSRIs inhibit bone development by affecting osteoblast maturation during embryonic development and MSC differentiation. These results highlight the need to further investigate the risks of SSRI use during pregnancy in exposing unborn babies to potential skeletal abnormalities. Molecular Psychiatry (2016) 21, 656–664; doi:10.1038/mp.2015.135; published online 8 September 2015

INTRODUCTION The safety of maternal selective serotonin reuptake inhibitors (SSRI) use during pregnancy is of increasing concern with recent studies suggesting associations between SSRIs and increased risk of persistent pulmonary hypertension, low birth weight, preterm birth, seizures and even infant death.1–4 Decreased birth length caused by SSRI use during pregnancy may suggest an effect on skeletal development.5,6 In adults, SSRI exposure has been shown to increase fracture risk, impair bone metabolism and decrease bone mineral density.7–10 We recently showed that the human bone-forming cells, osteoblasts, and the bone-resorbing cells, osteoclasts, express serotonin receptors and the serotonin transporter, the target of SSRI action, suggesting a possible mechanism for the effect of SSRIs on bone homeostasis.11 Furthermore, SSRIs can accumulate in the bone marrow for extended periods at concentrations much higher than those found in blood or the brain.12 Several SSRIs are currently considered as options for use for the treatment of depression during pregnancy, including citalopram and sertraline.13 Citalopram and sertraline account for ~ 70% of all SSRI prescriptions in the United States of America, including for pregnant

women.14 To date, an understanding of the impact of SSRIs on fetal bone development is lacking. The focus of this research, therefore, was to investigate effects on bone formation during embryonic development following exposure to SSRIs. Owing to the ethical and practical limitations of studying maternal SSRI use on fetal bone development in humans, our group has used the zebrafish (Danio rerio) as a model to study effects of SSRIs on embryonic bone formation. The zebrafish has been used as a vertebrate model organism for embryogenesis for over 30 years.15 The external and rapid development of the zebrafish makes it an excellent model for developmental biology, including bone development.16–20 Importantly, regulators of bone formation, cellular organization and bone homeostasis are highly conserved between zebrafish and humans.21,22 It is also known that 84% of human disease-related genes are expressed in zebrafish,23 and these features have allowed us to visualize bone formation in vivo following SSRI exposure. To complement our embryonic in vivo studies, we utilized an in vitro model of human osteoblast differentiation from early precursor cells that employs human adipose tissue-derived mesenchymal stem cells (AT-MSCs). These multipotent precursor cells provide an excellent source of osteoblast progenitor cells that can differentiate into mature

1 Metabolic Genetic Diseases Laboratory, Metabolic Research Unit, School of Medicine, Deakin University, Geelong, VIC, Australia; 2IMPACT and MMR Strategic Research Centres, School of Medicine, Deakin University, Geelong, VIC, Australia; 3Barwon Biomedical Research, University Hospital, Geelong, VIC, Australia; 4Orygen, The National Centre of Excellence in Youth Mental Health and the Centre for Youth Mental Health, Department of Psychiatry, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia and 5Department of Medicine, Northwest Academic Centre, The University of Melbourne, St Albans, VIC, Australia. Correspondence: Dr Y Gibert, IMPACT and MMR Strategic Research Centres, School of Medicine, Deakin University, 75 Pidgons Road, Waurn Ponds, Geelong, VIC 3217, Australia. E-mail: [email protected] 6 These authors contributed equally to this work. 7 These authors are co-senior authors. Received 18 September 2014; revised 15 May 2015; accepted 14 July 2015; published online 8 September 2015

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657 osteoblasts. To address the knowledge gap regarding the effects of SSRI exposure on early bone formation, we have investigated their actions in both an in vivo model of embryogenesis, as well as an in vitro human osteoblast model. MATERIALS AND METHODS Ethics statement All zebrafish studies were approved by the Deakin University Animal Welfare Committee (81-2011). Human adipose tissue samples were obtained with informed, written consent from healthy donors and all protocols were approved by the Barwon Health Human Research Ethics Committee (05–69).

resuspended and filtered through a 100-μm cell strainer to remove remaining tissue debris. Cells were pelleted by centrifugation and seeded at 1 × 106 in tissue culture flasks in basal medium and incubated at 37 °C in a humidified atmosphere with 5% CO2. Cells were passaged by treatment with 0.025% trypsin/EDTA in phosphate-buffered saline and diluted 1:10 in DMEM/FBS. MSCs were employed in assays after five passages.

Differentiation of MSC in medium containing osteogenic factors MSC (104 cells per well) were seeded in 6-mm diameter culture wells in DMEM/FBS and cultured overnight. For MSC differentiation, cells were then cultured in osteogenic medium (DMEM/FBS containing 100 nM dexamethasone, 10 mM β-glycerophosphate and 100 mM ascorbate-2-phosphate) in the absence or presence of citalopram or sertraline, and assessed for alkaline phosphatase (ALP) activity.

Animal husbandry Zebrafish were reared and staged at 28.5 °C according to Kimmel et al.24

Pharmacological treatment Citalopram (Sigma-Aldrich, St Louis, MO, USA) was stored at − 20 °C in a stock concentration of 24.7 mM dissolved in ethanol. Sertraline (SigmaAldrich) was stored at − 20 °C in a stock concentration of 29.2 mM dissolved in dimethyl sulphoxide. For treatment, embryos were placed in 50-ml tubes containing 25 ml of E3 embryo medium, supplemented with 0.003% phenyl-thiourea to prevent pigmentation.25 SSRIs were added directly to the embryonic medium and embryos were allowed to develop in an incubator at 28.5 °C.

Bone, cartilage and apoptosis staining Alizarin red skeletal staining was performed as previously described by Walker et al.26 Von Kossa staining was performed as described by Felber et al.27 Live embryos were stained for apoptotic cells with the vital dye acridine orange (Sigma-Aldrich) as described by Sassi-Messai et al.28 Cleaved caspase 3 labeling was performed with BD Pharmigen caspase 3, Active Form antibody (BD Biosciences, North Ryde, Sydney, Australia) and Donkey anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor 488 (Life Technologies, Mulgrave, Melbourne, Australia) as described by Sorrells et al.29 Alcian blue stain was adapted from Schilling et al.30 in which a 0.1% alcian blue solution was prepared in 0.37% HCl/70% ethanol.

Whole-mount in situ hybridization Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline overnight at 4 °C and then transferred and stored in 100% methanol. Whole-mount in situ hybridization using digoxigenin-labeled riboprobes was performed as described in Thisse and Thisse.31 The following probes were used: runx2a,32 runx2b,32 osterix,33 col10a1,34 sparc,35 spp1,35 sox9a,32 slc6a4a36 and caspase 3 (primers sequences: forward, 5ʹ-GCACTGACGT AGATGCAGGA-3ʹ; reverse, 5ʹ-GCCAATTGCTACCAGATTCC-3ʹ). Images were taken using the Axioskop 2 Imager (Zeiss, Carl Zeiss AG, Oberkochen, Germany) and processed with the Adobe Photoshop software (San Jose, CA, USA).

Cell media and reagents Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin solutions, paraformaldehyde, collagenase type-1, p-nitrophenylphosphate, p-nitrophenyl, diethanolamine, dexamethasone and dimethyl sulphoxide were purchased from Sigma-Aldrich. β-glycerophosphate disodium salt was purchased from Merck Millipore (Kilsyth, Victoria, Australia). L-Ascorbic acid phosphate was purchased from NovaChem Pty (Melbourne, Victoria, Australia). Non-essential amino acids (100 × ) and fetal bovine serum (FBS) were purchased from Bovogen (Melbourne, Victoria, Australia). All other reagents were of analytical grade.

Isolation and culture of adipose tissue-derived MSC Human adipose tissue was collected from elective abdominoplasty surgery. To isolate MSC, tissue was teased from blood vessels, minced with a scalpel blade and digested for 30–45 min with 0.075% collagenase at 37 °C with gentle agitation. Enzyme activity was neutralized with basal medium (DMEM containing 10% FBS, 50 U ml − 1 penicillin and 50 mg ml − 1 streptomycin) and the cells were centrifuged at 1200 g for 10 min, © 2016 Macmillan Publishers Limited

ALP activity assay To determine cellular ALP activity, cells were lysed in 0.1% Triton X-100 for 30 min at room temperature. A pre-warmed solution containing 10 mg ml − 1 p-nitrophenylphosphate in 10% v/v diethanolamine buffer containing 0.5 mM MgCl2 (pH 9.8) was then added to the lysates and optical density of samples was assessed using a Tecan Genios Pro photospectrometer, at 410 nm at 2.5 min intervals for 30 min. Results were converted to standard international units, equivalent to the conversion by ALP of 1 mM of pNPP to p-nitrophenyl (pNP) per minute. A standard curve was generated by serially diluting 1 mM pNP in diethanolamine buffer and data presented as relative standard international unit.

Measurement of cell viability/apoptosis Cell viability and apoptosis were assessed by propidium iodide (PI) staining and Annexin V (ANV) measurement, respectively. Previously untreated MSCs (4 × 105) were freshly prepared and then treated with citalopram or sertraline for 48 h. Cells were dissociated by trypsin digest (0.025%), recovered for 30 min and then washed in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and stained with a combination of ANV–FITC (5 μM per tube) and PI (10 μM) for 15 min in the dark. A further 400 μl of binding buffer was added to each sample and the percentage of viable (ANV − /PI − ) and apoptotic cells (ANV+/PI − ) was determined using flow cytometry analysis (FACSCalibur, CELLQuest software, BD Biosciences, Franklin Lakes, NJ, USA).

Real-time RT-PCR analysis Total RNA was isolated by lysing cells in Trizol and using the illustra RNAspin Mini Kit (GE Healthcare, Melbourne, Victoria, Australia) and reverse-transcribed using the Superscript III First Strand Synthesis SuperMix system (Life Technologies) as per the manufacturer’s instructions. For each SSRI dose/time point, mRNA was extracted separately from three wells of a 12-well plate and the real-time PCR was performed in duplicate. To quantify the expression of human osteoblastic genes (Runx2, ALP, Sparc and Spp1), human apoptotic genes (Bax and Caspase 3) and the zebrafish apoptotic gene (caspase 3), we employed quantitative real-time PCR (qRT-PCR) analysis of the cDNA in a 7500 Fast Real-Time PCR System (Applied Biosystems), using TaqMan Gene Expression Assays (Applied Biosystems, Hs00231692 (Runx2), Hs01029144 (ALP), Hs00234160 (Sparc), Hs00959010 (Spp1); Hs01016552 (Bax), Hs00234387 (Caspase 3) and zebrafish caspase 3.37 Relative gene expression units were determined using the formula 2 − ΔCtx1000, where ΔCt values represent the difference between the Ct of the gene of interest and Beta-actin (amplified using Taqman chemistry with forward primer (5′-GACAGGATGCAGAAGGA GATTACT-3’), reverse primer (5′-TGATCCACATCTGCTGGAAGGT-3′) and probe (5′(FAM)-ATCATTGCTCCTCCTGAGCGCAAGTACTC-(TAMRA)-3′).

Area measurement and statistical analysis Pictures of stained zebrafish embryos were mounted on a slide in a ventral orientation using an Axioskop 2 Imager (Zeiss). The area of stain was measured using Cell Sens Dimension (Olympus, Tokyo, Japan). Statistical analysis was performed using IBM SPSS Statistics version 21 (Armonk, NY, USA). Comparisons were made with an independent samples t-test. For human samples, data are expressed as the mean ± s.e.m., where applicable. Differences between groups were determined by one-way analysis of variance, followed by Fisher’s multiple comparison test. Molecular Psychiatry (2016), 656 – 664

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RESULTS Sertraline and citalopram inhibit bone development To assess the effects of citalopram and sertraline on bone in vivo, zebrafish embryos were treated from 36 h post fertilization (hpf), a developmental stage during embryogenesis when bone formation begins,21 through to 130 hpf. At 130 hpf, zebrafish embryos still rely on nutrition from the oocyte-provided yolk, preventing possible differential nutritional effects on development, whereas bone mineralization progressed enough to observe any effects due to chemical exposure. To determine the dosage and toxicity profile of each drug, embryos were exposed to increasing incremental doses until toxic effects (smaller or misshapen bodies) were observed. Embryos were treated with 10, 20 and 50 μM citalopram and sertraline (data not shown). Both citalopram and sertraline treatments caused growth retardation in the embryos at 50 μM, whereas treatment at 20 μM was toxic for citalopramtreated embryos, but not sertraline-treated embryos (data not shown). Therefore, we chose a dose range of 5–15 μM for citalopram and 10–30 μM for sertraline (Figures 1c–h, k–p). Both citalopram and sertraline treatments decreased tissue mineralization in developing bones, observed by staining with the matrix calcification dye alizarin red, in a dose-dependent manner (Figures 1a–h, i–p). This inhibition of bone formation was notably present in the vertebral column (arrows) and, in the opercle, the posterior part of the operculum, a specified bone that covers and protects the gills in fish (arrowheads). Magnification of the opercle bone shows decreased calcification upon exposure to citalopram and sertraline at 15 and 30 μM, respectively, compared with control embryos (Figures 1q–t). Quantification of the amount of calcified tissue in the whole embryo was consistent with the effects observed in the opercle, with reductions in the total area of alizarin red staining at higher SSRI concentrations (Figures 1u, v). To assess bone mineralization under SSRI exposure, embryos were treated with 15 μM citalopram and 30 μM sertraline from 36 to 150 hpf (a time point more suitable for observing von Kossa staining) and stained with von Kossa stain to label matrix phosphate (Figures 1w, z). Both citalopram- and sertraline-treated embryos showed reduced mineralization compared with controls, notably in the opercle (Figures 1w–z, arrowheads and insets). Owing to the reduction of bone calcification and mineralization that we observed from citalopram and sertraline exposure, we monitored cartilage development in embryos exposed to the SSRIs. We were unable to detect changes in the expression of sox9a, a chondrogenic marker, or differences in cartilage morphology, labeled with alcian blue, a cartilage stain (Supplementary Figure 1A–P). Therefore, the effects of citalopram and sertraline are specific to bone development and are not chondrogenesis. As the mode of action for SSRIs is to inhibit proper functioning of the serotonin transporter, we performed whole-mount in situ hybridization to identify expression of the transporter in embryonic zebrafish bone. In zebrafish, there are two identified serotonin transporters, slc6a4a and slc6a4b.36 At 72 hpf, we detected expression of slc6a4a in the opercle (Figure 1a', b' arrowheads) and cleithrum (Figure 1c' bracket) bones. Expression

of slc6a4a was not detected in these areas at the early time points 50 and 62 hpf (data not shown). To further investigate the effects of citalopram and sertraline on bone development, we examined the expression of genes involved in early (runx2a and runx2b), intermediate (osterix, also called sp7) and mature (collagen 10a1, osteonectin (sparc) and osteopontin (spp1)) stages of osteoblast differentiation in fish. Zebrafish embryos were treated with SSRIs from 36 hpf, when pre-osteoblast cells begin expressing runx2b in forming skeletal condensations.21 We allowed the embryos to develop until 50 hpf and performed whole-mount in situ hybridization with runx2a and runx2b probes (n410). Citalopram and sertraline had no effect on runx2a or runx2b expression compared with control embryos, notably in the area of the forming opercle (Figures 2a–d, e–h arrowheads). The expression of osterix (osx), a later marker of osteoblast differentiation, was also unaffected in embryos treated from 36 to 62 hpf (Figures 2i–l, arrowheads, n410). Expression of collagen 10a1 (col10a1) which, in zebrafish, is first expressed from 63 hpf onwards in osteoblasts,21 was decreased in the area of the developing opercle (10/12 citalopram treated, 14/18 sertraline treated) in embryos exposed to SSRI treatment from 36 to 72 hpf (Figures 2m–p, arrowheads). The expression of two bone matrix proteins, sparc and spp1, was reduced in the opercle (n410, all treated embryos had reductions) in citalopram- and sertraline-treated embryos (Figures 2q–t, u–x, arrowheads). Therefore, citalopram and sertraline treatments appear only to affect the expression of genes present later in osteoblast differentiation. To determine whether SSRI treatment had a transient effect on bone development, only inhibiting late bone development, we treated embryos at an early time range, from 36 to 56 hpf. Embryos treated with citalopram and sertraline from 36 to 56 hpf showed no differences in alizarin red staining at 130 hpf compared with controls (Supplementary Figure 2A and D). In addition, embryos treated from 36 to 56 hpf showed no differences in the expression of col10a1, sparc and spp1 in the opercle at 72 hpf (Supplementary Figure 2 E–H,I-L,M-P, arrowheads). Therefore, the effects of citalopram and sertraline on bone development appear to result from the action of SSRIs during late osteoblast development. The effects of citalopram and sertraline on bone development are independent of apoptosis in zebrafish embryos As cell death could cause reduced mineralization, calcification and gene expression, we investigated necrosis and apoptosis in treated embryos. Bright-field images showed no evidence of tissue necrosis (opaque cells) from 15 μM citalopram and 30 μM sertraline treatments when embryos were exposed from 36 to 50 hpf (data not shown), 36 to 72 hpf (data not shown) and 36 to 96 hpf (Figures 3a–d). To assess apoptosis, zebrafish embryos were stained with acridine orange, a fluorescent dye marking apoptotic cells, following citalopram and sertraline exposure from 36 to 96 hpf (Figures 3e–h, arrowheads). There was no evidence of apoptosis occurring in areas of developing bone and no detectable increase in overall apoptosis. Acridine orange staining

Figure 1. Citalopram and sertraline exposure decreased bone calcification and mineralization during zebrafish embryogenesis. Embryos were treated with citalopram (c–h) or sertraline (k–p) from 36 to 130 h post fertilization (hpf ) and stained with alizarin red after fixation at 130 hpf. Embryos were treated with 5 μM (c and d), 10 μM (e and f) or 15 μM (g and h) citalopram and 10 μM (k and l), 20 μM (m and n) or 30 μM (o and p) sertraline. Citalopram- and sertraline-treated embryos showed a dose-dependent decrease in bone calcification, specifically in the opercle (arrowheads), compared with control siblings (a, b, i and j). The area of staining in (pixel)2 of the opercle was measured in control fish (q, close up of the opercle in r), citalopram-treated (s) and sertraline-treated embryos (t). Both citalopram and sertraline treatments showed a decrease in alizarin red staining compared with control fish. Total area of alizarin red staining was measured in citalopram- and sertraline-treated fish (u and v). Both treatments showed a dose-dependent decrease in area of alizarin red staining with increasing concentration. Values are shown as relative to control, *significantly different to control (Po 0.05). Von Kossa staining, to measure bone mineralization, of 15 μM citalopram (x) or 30 μM sertraline (z)-treated fish from 36 to 150 hpf showed reduced mineralization of the opercle (arrowheads, insets) compared with controls (w and y). Expression of the serotonin transporter, slc6a4a, is shown in wild-type embryos at 72 hpf (a'–c') located in the opercle (arrowheads) and cleithrum (bracket). Molecular Psychiatry (2016), 656 – 664

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659 of embryos treated from 36 to 50 hpf and 36 to 72 hpf also showed no increase in apoptosis (Supplementary Figure 3A–D, E–H). In addition to no change in acridine orange staining, expression of caspase 3 (a key caspase involved in apoptosis) did

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not change in embryos treated with citalopram or sertraline from 36 to 52 hpf or from 36 to 72 hpf (Figures 3i–p, blue arrowheads). qRT-PCR analyses from whole-embryo mRNA confirmed that caspase 3 mRNA levels were not increased in citalopram- or

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Figure 2. Citalopram and sertraline exposure affected the expression of only late zebrafish osteoblast markers. Whole-mount in situ hybridization was performed on embryos treated with 15 μM citalopram and 30 μM sertraline. Embryos treated from 36 to 56 h post fertilization (hpf ) (c, d, g and h) did not show a difference in runx2b or runx2a expression in the developing opercle (arrowhead) compared with controls (a, b, e and f). Embryos treated from 36 to 62 hpf (k and l) did not show a difference in osx expression compared with controls (i and j). col10a1 expression in embryos treated from 36 to 72 hpf (o and p) was reduced, prominently in the opercle (arrowheads), compared with controls (m and n). The expression of sparc (q–t) and spp1 (u–x) was reduced in embryos treated from 36 to 72 hpf.

sertraline-treated embryos (Figure 3q). We further evaluated apoptosis by using antibody labeling for cleaved caspase 3, the activated form of the caspase 3 protein. Embryos treated with citalopram and sertraline from 36 to 96 hpf showed no increase in cleaved caspase 3 labeling compared with controls (Figures 3r–u, arrows). Similarly, embryos treated from 36 to 50 hpf or 36 to 72 hpf did not show increases in cleaved caspase 3 labeling (Supplementary Figure 3 I-L,M-P). Therefore, cell death from apoptosis or necrosis does not appear to be the cause of decreased bone development. Osteoblast differentiation from human AT-MSCs is inhibited by citalopram and sertraline Both citalopram and sertraline decreased ALP activity, a marker of osteoblast lineage, in a dose-dependent manner in AT-MSC cultured in osteogenic media for 7 days (Figure 4 ai). Sertraline was more potent than citalopram, with decreases in ALP activity of 55 and 16% at 10 μM, respectively (Po 0.001). AT-MSCs were exposed to citalopram and sertraline at intervals of 0–7, 8–14 and 0–14 days and ALP activity was assessed at 14 days (Figure 4 aii). Sertraline-treated AT-MSCs showed decreased ALP activity when exposed from 8 to 14 and 0 to 14 days. Consistent with this finding, expression of ALP mRNA was reduced by 42% after 7 days and by 48% after 14 days of treatment with sertraline, and by 29% when treated with citalopram after 14 days (Figure 4 aiii, iv). There was a trend for decreased expression of Runx2 mRNA at 7 days of treatment; however, this only reached significance in the case of sertraline at 14 days, with a 53% decrease (Figure 4 av, vi). In addition, Sparc expression was significantly decreased at 13 days when AT-MSCs were treated with sertraline from 0 to 7 and 0 to 13 days (Figure 4 avii). AT-MSCs treated from 8 to 13 days showed a decrease in Sparc expression approaching significance (P = 0.054; Figure 4 avii). Furthermore, the expression of Spp1 was decreased Molecular Psychiatry (2016), 656 – 664

at 13 days in AT-MSCs that were exposed to sertraline from 0 to 7, 8 to 13 and 0 to 13 days (Figure 4 aviii). Therefore, in the human model, sertraline shows a higher capacity to inhibit osteoblast differentiation than citalopram, although AT-MSCs exposed to citalopram did show modest decreases in ALP activity and expression at 7 and 14 days, respectively. To determine the role of apoptosis in decreasing osteoblast differentiation, Annexin V staining following 48 h of exposure was performed. Sertraline treatment induced a minor, dose-dependent increase in the percentage of apoptotic cells, whereas citalopram had no effect (Figure 4 bi). Furthermore, neither sertraline nor citalopram treatment affected expression levels of apoptotic markers Bax and Caspase 3 compared with control levels at 14 days (Figure 4 Bii–iv), whereas a significant increase in Bax mRNA was observed after 7 days of sertraline exposure (Figure 4 bii). DISCUSSION SSRI use during pregnancy The central focus of these experiments was to study the effects of SSRIs specifically during embryonic bone formation. Depression is common during pregnancy, with a study showing the prevalence of depressive symptoms to be 17%, with another reporting that 3.8% of women take an SSRI during pregnancy.38,39 There is existing evidence suggesting that SSRI use by pregnant mothers is associated with birth defects. Chambers et al. showed an association between persistent pulmonary hypertension of the newborn and SSRI exposure after completion of the 20th week of gestation.1 Wen et al. examined a retrospective cohort of 972 pregnant women who had received at least one SSRI prescription within the year before delivery compared with a cohort of 3878 women who had not taken SSRIs.2 The researchers found that infants from mothers who had taken SSRIs were at a higher risk for low birth weight, preterm birth, seizures and even infant death.2 © 2016 Macmillan Publishers Limited

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Figure 3. Citalopram and sertraline did not affect apoptosis in the developing zebrafish. Embryos were treated with 15 μM citalopram or 30 μM sertraline. Bright-field images of embryos treated from 36 to 96 h post fertilization (hpf ) showed no increase in cell necrosis (a–d), which would be observed as opaque tissue. Embryos treated from 36 to 96 hpf were stained with acridine orange dye to label apoptotic cells (e–h, arrowheads) and showed comparable levels of labeled cells with controls (open arrowheads label neuromasts, not to be confused as apoptotic cells). Whole-mount in situ hybridization was performed with a caspase 3 probe on embryos treated from 36 to 52 hpf (k and l) and 36–72 hpf (o and p). Treated embryos showed no difference in expression (blue arrowheads) compared with controls (i, j, m and n). Quantitative real-time PCR shows that there was no significant difference in relative gene expression of caspase 3 in treated embryos compared with the controls (q). Cleaved caspase 3 antibody labeling of treated embryos (s and u, arrows) was consistent with levels seen in control embryos (r and t).

Furthermore, Alwan et al.40 showed an association between maternal SSRI use and infant anencephaly, craniosynostosis and omphalocele.40 These findings have raised concerns regarding the use of SSRIs during pregnancy, reflected by these data. SSRIs and bone formation Although evident in the elderly, osteoporosis is more accurately a disorder of the lifespan, beginning with variable rates of bone formation in youth, and driven by variable rates of bone loss in adulthood. Although there has been a rise in research examining the effects of SSRIs on the bone, few studies have looked at the effects on bone during development. One study by Dubnov-Raz et al. analyzed the bone density of infants between 1 and 4 days of life whose mothers had been using SSRIs during gestation.6 Although a difference in the bone density of infants exposed to SSRIs was not evident, these infants were shorter and had smaller heads, indicating a possible defect in skeletal growth. A limitation of that study was that ultrasound, rather than the gold standard, dual-energy X-ray absorptiometry, was used to determine bone mineral density in order to avoid potential negative health impacts of radiation on the children. Our study was able to observe the initial stages of bone formation in a developing organism in vivo. Our data indicated that both citalopram and sertraline decreased bone mineralization during zebrafish embryogenesis and osteoblast activity during differentiation. It is reasonable to speculate that these effects could hold true in human fetuses, which could present future health risks. A negative effect on bone maturation in the fetus could have lifelong implications as it has been shown that skeletal growth in adulthood is influenced by intrauterine development.41 Our results © 2016 Macmillan Publishers Limited

showed that SSRIs appear to affect the later stages of osteoblast differentiation, as only expression of later zebrafish osteoblast markers, col10a1, sparc and spp1, were reduced by SSRI's exposure (Figure 4c). Interestingly, the onset of expression of these genes coincides with the initiation of the expression of the serotonin transporter slc6a4a in bone tissue during zebrafish embryogenesis (Figures 1a'–c'). It is possible that the presence of the transporter is required for the inhibitory actions of the SSRIs to manifest and support the observation that early osteoblast progression is unaffected by SSRIs. Therefore, on the basis of these results we can hypothesize that maternal SSRI use may put the fetus at risk of bone malformations in the later stages of pregnancy, depending on the timing of expression of the serotonin transporter in human bone development, when fetal osteoblasts are maturing. Concentration of SSRIs and bone formation The concentrations of citalopram and sertraline administered to the zebrafish embryos in this study were higher than the recommended maximum dosage prescribed for use in humans, which is up to 40 mg per day for citalopram and up to 200 mg per day for sertraline.42,43 At this dosage, the citalopram serum levels can be as high as 250 ng ml − 1 (equivalent to a concentration of 0.8 μM), whereas sertraline serum levels can be as high as 200 ng ml − 1 (equivalent to a concentration of 0.7 μM).43,44 Whereas both of these concentrations are still much lower than the concentrations used in our experiments, a previous study has shown that the SSRI, fluvoxamine, can accumulate in the bone marrow at much higher concentrations than are present in circulating serum.12 Fluvoxamine levels were shown to be as high Molecular Psychiatry (2016), 656 – 664

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Figure 4. Effect of citalopram and sertraline on osteoblastic maturation and induction of apoptosis in human MSC. Human MSCs were cultured in osteogenic media in the presence of citalopram and sertraline. (ai, ii) alkaline phosphatase (ALP) activity at 7 and 14 days; (iii, iv) ALP gene expression at 7 and 14 days; (v, vi) Runx2 gene expression at 7 and 14 days; (vii) Sparc gene expression at 13 days; (viii) Spp1 gene expression at 13 days. (bi) Percentage of apoptotic cells (Annexin V+ve) after 48 h of treatment and assessment using flow cytometry; (ii, iii) Bax gene expression at 7 and 14 days; (iv, v) Caspase 3 gene expression at 7 and 14 days. Data are expressed as mean ± s.e.m. where applicable. RT-PCR values are shown relative to control values. Statistical significance is indicated thus: citalopram different to control, ★; sertraline different to control, #; citalopram different to sertraline, μ; Po 0.05. (c) Model of SSRI action on bone formation during zebrafish development. MSC, mesenchymal stem cell; RT-PCR, real-time PCR; SSRI, selective serotonin reuptake inhibitor.

as 80 μM in the bone marrow of human patients, which is more than five times the concentration of citalopram and more than 2.5 times the concentration of sertraline used to treat zebrafish embryos in this study. Furthermore, fluvoxamine remained at an elevated level, above 60 μM, in the bone marrow even as the circulating level fell below detectable levels following cessation of treatment. These data demonstrate that SSRIs have the capacity to bioaccumulate in the bone marrow. Therefore, SSRI use, even transiently during pregnancy, might have deleterious effects on bone formation, as SSRIs remain at high levels in the bone marrow even after their levels have dropped in blood. As the bone marrow is essential for bone formation via osteoblast activity, it is tempting to hypothesize that SSRI use by a pregnant mother during the time that the bone marrow has developed in the fetus may affect osteoblast activity and bone mineralization. SSRI and apoptosis We recently reported that most SSRIs (sertraline, fluvoxamine, fluoxetine and paroxetine) induced apoptosis in bone cells, whereas Molecular Psychiatry (2016), 656 – 664

citalopram did not.11 These findings are in the context of previous reports of citalopram acting as an anti-apoptotic agent in vivo in brain structures and that sertraline was a known inducer of apoptosis.45–47 On the basis of these results, it was expected that citalopram would not induce apoptosis; however, it was anticipated that sertraline might, thus preventing proper bone mineralization. However, this was not observed in our in vivo model, as both sertraline and citalopram exposure did not induce apoptosis during embryogenesis. In comparison, during bone differentiation from human MSCs, a high level of sertraline exposure led to a small increase in apoptosis after 48-h exposure and an increase in expression of the apoptosis regulator Bax after 7-day exposure, whereas after 14-day exposure Bax expression was unchanged from control. However, the apoptotic changes seen at the earlier time points were not accompanied by alterations to expression of Caspase 3 and may have been transient. In the human model of osteoblast differentiation from MSCs, we observed a strong reduction in ALP expression after 14-day treatment of both sertraline and citalopram. Furthermore, we observed a reduction in Runx2 expression at 14 days of exposure in sertraline-treated cells © 2016 Macmillan Publishers Limited

Sertraline and citalopram in bone formation D Fraher et al

663 and a trend of reduction in citalopram-treated cells. These reductions at 14 days of treatment cannot be caused by apoptosis as neither citalopram- nor sertraline-treated cells exhibited a higher level of Bax or Caspase 3 after 14 days of exposure. Furthermore, citalopram-treated cells did not exhibit an increase in Bax expression but nevertheless displayed a strong reduction in ALP mRNA after 14 days of differentiation. We therefore conclude from these data that during zebrafish embryogenesis and human osteoblast differentiation, apoptosis is not the biological process by which decreases in bone mineralization and in ALP activity occur. During zebrafish embryogenesis, osteoblasts are first detected by gene expression at around 36 hpf,21 whereas bone mineralization is first detected at around 96 hpf.48 During this time, osteoblasts rapidly mineralize bone to produce the mature skeleton. The goal of this rapid production is to form a supportive skeleton fast enough to provide rigidity for the developing organism. Apoptosis at these developmental stages would be counterproductive to the skeletal building process. Therefore, it is possible that, although SSRIs can induce apoptosis in adult osteoblasts,11 the developing bone cells are more resistant to apoptosis – a feature previously reported in other stem/progenitor cells this apoptotic stimulus is overcome in developing bone cells.49 This could have potentially prevented the SSRIs from inducing apoptosis in our models of bone formation during early embryogenesis. CONCLUSION To our knowledge, this is the first study to demonstrate that SSRIs directly influence bone formation during vertebrate embryonic development and in the differentiation of MSCs into osteoblasts. Our results confirm previous studies suggesting that sertraline may have higher bone toxicity than citalopram, especially during human MSC differentiation into osteoblasts. Given the high prevalence of bone-related disease,50 the use of SSRIs to treat psychiatric disorders during pregnancy, and the knowledge that SSRI agents affect bone formation, our study has overt clinical implications. If supported by human clinical and epidemiological data, it may be time to re-evaluate the current guidelines for SSRI prescriptions, incorporating recommendations for SSRI use during pregnancy in order to prevent potential bone defects in babies born to mothers experiencing mood disorders. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGMENTS We thank the staff members of the Deakin University zebrafish facility for providing excellent husbandry care. YG is supported by funding from the Molecular and Medical Research Strategic Centre at Deakin University. MB is supported by a NHMRC Senior Principal Research Fellowship (1059660). LJW is supported by a NHMRC Career development Fellowship (1064272).

REFERENCES 1 Chambers CD, Hernandez-Diaz S, Van Marter LJ, Werler MM, Louik C, Jones KL et al. Selective serotonin-reuptake inhibitors and risk of persistent pulmonary hypertension of the newborn. N Engl J Med 2006; 354: 579–587. 2 Wen SW, Yang Q, Garner P, Fraser W, Olatunbosun O, Nimrod C et al. Selective serotonin reuptake inhibitors and adverse pregnancy outcomes. Am J Obstet Gynecol 2006; 194: 961–966. 3 Andrade SE, Raebel MA, Brown J, Lane K, Livingston J, Boudreau D et al. Use of antidepressant medications during pregnancy: a multisite study. Am J Obstet Gynecol 2008; 198: 194 e1–194 e5. 4 Laine K, Heikkinen T, Ekblad U, Kero P. Effects of exposure to selective serotonin reuptake inhibitors during pregnancy on serotonergic symptoms in newborns and cord blood monoamine and prolactin concentrations. Arch Gen Psychiatry 2003; 60: 720–726.

© 2016 Macmillan Publishers Limited

5 Davidson S, Prokonov D, Taler M, Maayan R, Harell D, Gil-Ad I et al. Effect of exposure to selective serotonin reuptake inhibitors in utero on fetal growth: potential role for the IGF-I and HPA axes. Pediatr Res 2009; 65: 236–241. 6 Dubnov-Raz G, Hemilä H, Vurembrand Y, Kuint J, Maayan-Metzger A. Maternal use of selective serotonin reuptake inhibitors during pregnancy and neonatal bone density. Early Hum Dev 2012; 88: 191–194. 7 Chau K, Atkinson SA, Taylor VH. Are selective serotonin reuptake inhibitors a secondary cause of low bone density? J Osteop 2012; 2012: 323061. 8 Haney EM, Chan BK, Diem SJ, Ensrud KE, Cauley JA, Barrett-Connor E et al. Association of low bone mineral density with selective serotonin reuptake inhibitor use by older men. Arch Int Med 2007; 167: 1246–1251. 9 Richards JB, Papaioannou A, Adachi JD, Joseph L, Whitson HE, Prior JC et al. Effect of selective serotonin reuptake inhibitors on the risk of fracture. Arch Int Med 2007; 167: 188–194. 10 Williams LJ, Henry MJ, Berk M, Dodd S, Jacka FN, Kotowicz MA et al. Selective serotonin reuptake inhibitor use and bone mineral density in women with a history of depression. Int Clin Psychopharmacol 2008; 23: 84–87. 11 Hodge JM, Wang Y, Berk M, Collier FM, Fernandes TJ, Constable MJ et al. Selective serotonin reuptake inhibitors inhibit human osteoclast and osteoblast formation and function. Biol Psychiatry 2013; 74: 32–39. 12 Bolo N, Hode Y, Macher J-P. Long-term sequestration of fluorinated compounds in tissues after fluvoxamine or fluoxetine treatment: a fluorine magnetic resonance spectroscopy study in vivo. Magn Reson Mater Phys Biol Med 2004; 16: 268–276. 13 MayoClinicStaff. Antidepressants: Safe During Pregnancy?: Mayo Clinic; 2012 (cited 4 August 2014). Available from http://www.mayoclinic.org/healthy-living/ pregnancy-week-by-week/in-depth/antidepressants/art-20046420. 14 Grohol JM. Top 25 Psychiatric Medication Prescriptions for 2013: Psych Central; 2014 (cited 2014 24 July 2014). Available from http://psychcentral.com/lib/top-25psychiatric-medication-prescriptions-for-2013/00019543. 15 Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim into view. Nat Rev Genet 2007; 8: 353–367. 16 Zon LI. Zebrafish: a new model for human disease. Genome Res 1999; 9: 99–100. 17 Spoorendonk KM, Peterson-Maduro J, Renn J, Trowe T, Kranenbarg S, Winkler C et al. Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton. Development 2008; 135: 3765–3774. 18 Knopf F, Hammond C, Chekuru A, Kurth T, Hans S, Weber CW et al. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev Cell 2011; 20: 713–724. 19 Fisher S, Halpern ME. Patterning the zebrafish axial skeleton requires early chordin function. Nat Genet 1999; 23: 442–446. 20 Barrett R, Chappell C, Quick M, Fleming A. A rapid, high content, in vivo model of glucocorticoid‐induced osteoporosis. Biotechnol J 2006; 1: 651–655. 21 Li N, Felber K, Elks P, Croucher P, Roehl HH. Tracking gene expression during zebrafish osteoblast differentiation. Dev Dyn 2009; 238: 459–466. 22 Flores MV, Tsang VWK, Hu W, Kalev-Zylinska M, Postlethwait J, Crosier P et al. Duplicate zebrafish runx2 orthologues are expressed in developing skeletal elements. Gene Expr Patterns 2004; 4: 573–581. 23 Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 2013; 496: 498–503. 24 Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn 1995; 203: 253–310. 25 Geisler R. Zebrafish: A Practical Approach (The Practical Approach Series, 261). Oxford Univ Press: New York, NY, USA, 2002, pp 175–212. 26 Walker M, Kimmel C. A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech Histochem 2007; 82: 23–28. 27 Felber K, Croucher P, Roehl HH. Hedgehog signalling is required for perichondral osteoblast differentiation in zebrafish. Mech Dev 2011; 128: 141–152. 28 Sassi-Messai S, Gibert Y, Bernard L, Nishio S-I, Lagneau KFF, Molina J et al. The phytoestrogen genistein affects zebrafish development through two different pathways. PLoS One 2009; 4: e4935. 29 Sorrells S, Toruno C, Stewart RA, Jette C. Analysis of apoptosis in zebrafish embryos by whole-mount immunofluorescence to detect activated Caspase 3. J Vis Exp 2013; 82: e51060. 30 Schilling TF, Piotrowski T, Grandel H, Brand M, Heisenberg C-P, Jiang Y-J et al. Jaw and branchial arch mutants in zebrafish I: branchial arches. Development 1996; 123: 329–344. 31 Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 2007; 3: 59–69. 32 Eames BF, Amores A, Yan Y-L, Postlethwait JH. Evolution of the osteoblast: skeletogenesis in gar and zebrafish. BMC. Evol Biol 2012; 12: 27. 33 Hammond CL, Schulte-Merker S. Two populations of endochondral osteoblasts with differential sensitivity to Hedgehog signalling. Development 2009; 136: 3991–4000.

Molecular Psychiatry (2016), 656 – 664

Sertraline and citalopram in bone formation D Fraher et al

664 34 Albertson RC, Yan Y-L, Titus TA, Pisano E, Vacchi M, Yelick PC et al. Molecular pedomorphism underlies craniofacial skeletal evolution in Antarctic notothenioid fishes. BMC. Evol Biol 2010; 10: 4. 35 Laue K, Pogoda H-M, Daniel PB, van Haeringen A, Alanay Y, von Ameln S et al. Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid. Am J Hum Genet 2011; 89: 595–606. 36 Norton WH, Folchert A, Bally‐Cuif L. Comparative analysis of serotonin receptor (HTR1A/HTR1B families) and transporter (slc6a4a/b) gene expression in the zebrafish brain. J Comp Neurol 2008; 511: 521–542. 37 Mei J, Zhang Q-Y, Li Z, Lin S, Gui J-F. C1q-like inhibits p53-mediated apoptosis and controls normal hematopoiesis during zebrafish embryogenesis. Dev Biol 2008; 319: 273–284. 38 Domar A, Moragianni V, Ryley D, Urato A. The risks of selective serotonin reuptake inhibitor use in infertile women: a review of the impact on fertility, pregnancy, neonatal health and beyond. Hum Reprod 2013; 28: 160–171. 39 Josefsson A, Berg G, Nordin C, Sydsjö G. Prevalence of depressive symptoms in late pregnancy and postpartum. Acta Obstetr Gynecol Scand 2001; 80: 251–255. 40 Alwan S, Reefhuis J, Rasmussen SA, Olney RS, Friedman JM. Use of selective serotonin-reuptake inhibitors in pregnancy and the risk of birth defects. N Engl J Med 2007; 356: 2684–2692. 41 Cooper C, Fall C, Egger P, Hobbs R, Eastell R, Barker D. Growth in infancy and bone mass in later life. Ann Rheum Dis 1997; 56: 17–21. 42 FDA. FDA Drug Safety Communication: Revised Recommendations for Celexa (Citalopram Hydrobromide) Related to a Potential Risk of Abnormal Heart Rhythms with High Doses: U.S. Food and Drug Administration; 2012 (cited 3 August 2014). Available fromhttp://www.fda.gov/drugs/drugsafety/ucm297391.htm.

43 UniversityofIowa. Sertraline (Zoloft) Drug Level The University of Iowa Department of Pathology Laboratory Services Handbook: The University of Iowa Health Care; 2013 (cited 3 August 2014). Available fromhttps://www.healthcare.uiowa. edu/path_handbook/handbook/test2628.html. 44 MayoClinic. Test Catalog: Citalopram, Serum: Mayo Medical Laboratories; 2014 (cited 1 August 2014). Available from http://www.mayomedicallaboratories.com/ test-catalog/Clinical+and+Interpretive/83730. 45 Kosten TA, Galloway MP, Duman RS, Russell DS, D'Sa C. Repeated unpredictable stress and antidepressants differentially regulate expression of the bcl-2 family of apoptotic genes in rat cortical, hippocampal, and limbic brain structures. Neuropsychopharmacology 2007; 33: 1545–1558. 46 Murray F, Hutson PH. Hippocampal Bcl-2 expression is selectively increased following chronic but not acute treatment with antidepressants, 5-HT1A or 5-HT 2C/2B receptor antagonists. Eur J Pharmacol 2007; 569: 41–47. 47 Chen S, Xuan J, Wan L, Lin H, Couch L, Mei N et al. Sertraline, an antidepressant, induces apoptosis in hepatic cells through the mitogen-activated protein kinase pathway. Toxicol Sci 2014; 137: 404–415. 48 Seritrakul P, Samarut E, Lama TT, Gibert Y, Laudet V, Jackman WR. Retinoic acid expands the evolutionarily reduced dentition of zebrafish. FASEB J 2012; 26: 5014–5024. 49 Szegezdi E, O’Reilly A, Davy Y, Vawda R, Taylor DL, Murphy M et al. Stem cells are resistant to TRAIL receptor‐mediated apoptosis. J Cell Mol Med 2009; 13: 4409–4414. 50 Health UDo, Services H. Bone Health and Osteoporosis: a Report of the Surgeon General. US Department of Health and Human Services, Office of the Surgeon General: Rockville, MD, USA, 2004.

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