Camille C Savary

Nessrine Bellamri

Claudie Morzadec

Sophie Langouët

Valérie Lecureur

Laurent Vernhet
email: laurent.vernhet@univ-rennes1.fr

Long term exposure to environmental concentrations of Diesel exhaust particles does not impact the phenotype of human bronchial epithelial cells

Keywords: diesel exhaust particles, PAH: polycylic aromatic hydrocarbons, AhR: aryl hydrocarbon receptor, CYP: cytochrome P450, ROS: reactive oxygen species, B2B: Beas-2B, IL: Interleukin, SRM: Standard Reference Material, DAPI: 4′, 6-diamidino-2-phenylindole, TGF-β1: Tumor growth factor-β1, BEGM: Bronchial epithelial growth medium, -H2AX: phospho(ser139)-Histone 2AX, Cq: quantification cycle, dichlorodihydrofluorescein-diacetate, DCF: dichlorofluoroscein, MMC: Mitomycin C, NQO1: NADPH quinone oxidoreductase 1, EMT: epithelial-mesenchymal transition, B diesel exhaust particles, bronchial epithelial cells, toxicity, chronic exposure

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Introduction

Diesel engine exhausts (DEE) strongly contribute to air pollution in most industrial cities over the world. Chronic exposures to DEE induce irritations of the respiratory tract and can promote the development of respiratory diseases such as asthma, chronic obstructive pulmonary disease and cancers [START_REF] Steiner | Diesel exhaust: current knowledge of adverse effects and underlying cellular mechanisms[END_REF]. In 2012, based on the results of epidemiological studies, the International Agency for Research on Cancer has concluded that sufficient evidence associate DEE exposure to lung cancer and has thus classified DEE as carcinogenic for humans ("IARC," 2012).

DEE is a complex mixture of gas and diesel exhaust particles (DEP) consisting of an aggregated core of ultrafine and fine carbon particles that are mainly coated with polycyclic aromatic hydrocarbons (PAH) and nitro-PAH [START_REF] Steiner | Diesel exhaust: current knowledge of adverse effects and underlying cellular mechanisms[END_REF]. Following inhalation, these hydrocarbons can desorb from particles and induce toxicity on the surrounding lung epithelial cells [START_REF] Gerde | The rapid alveolar absorption of diesel soot-adsorbed benzo[a]pyrene: bioavailability, metabolism and dosimetry of an inhaled particle-borne carcinogen[END_REF]. Particularly, the carcinogenic effects of DEP mainly result from the ability of PAH and nitro-PAH to bind to and activate the arylhydrocarbon receptor (AhR). Once activated, the ligand/AhR complex translocates into the nuclei, dimerizes with the AhR nuclear translocator Arnt and binds to the xenobiotic responsive elements of many gene promoters [START_REF] Moorthy | Polycyclic aromatic hydrocarbons: from metabolism to lung cancer[END_REF]. AhR activation notably up-regulates the expression of the cytochrome P450 (CYP) 1A1 and 1B1, two metabolizing enzymatic complexes which bio-activate PAH and nitro-PAH into electrophilic and genotoxic metabolites [START_REF] Moorthy | Polycyclic aromatic hydrocarbons: from metabolism to lung cancer[END_REF]. In vivo and in vitro experimental studies suggest that DEP also induce carcinogenic effects by increasing the production of reactive oxygen species (ROS) that cause DNA damages and/or by stimulating the expression of pro-inflammatory cytokines and growth factors in lung epithelial cells [START_REF] Steiner | Diesel exhaust: current knowledge of adverse effects and underlying cellular mechanisms[END_REF].

Most in vitro studies reporting the toxic effects of DEP on lung epithelial cells have been performed using short exposures, lasting from 6 h to 72 h, and DEP concentrations ranging from 10 to 100 µg/ml (approximately 2 to 20 µg/cm 2 ) [START_REF] Jardim | Disruption of microRNA expression in human airway cells by diesel exhaust particles is linked to tumorigenesisassociated pathways[END_REF]Cao et al., 2010;[START_REF] Totlandsdal | Differential effects of the particle core and organic extract of diesel exhaust particles[END_REF]. [START_REF] Li | Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects[END_REF] have calculated that an exposure to ambient particulate matter levels of 79 µg/m 3 over 24 h in Rubidoux, a highly polluted site in California, leads to a particle mass deposition of 2.3 to 0.05 µg/cm 2 from tracheobronchial to alveolar regions, respectively [START_REF] Li | Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects[END_REF]. Although it seems very difficult to precisely reconcile in vitro and in vivo particle dosimetry, it thus appears that DEP concentrations used in experimental studies are generally higher than the levels to which humans are exposed. Surprisingly, no in vitro study has assessed the toxicity of low DEP levels on chronically exposed lung epithelial cells.

The fact that primary human lung epithelial cells are very difficult to culture probably explains this lack of experimental studies reporting long term toxicity of DEP. By comparing human lung cell models, [START_REF] Courcot | Xenobiotic metabolism and disposition in human lung cell models: comparison with in vivo expression profiles[END_REF] have shown that the human bronchial epithelial BEAS-2B (B2B) cells display the highest homology in gene expression pattern with primary cells. These cells may thus constitute an interesting cell line to assess chemical toxicity [START_REF] Courcot | Xenobiotic metabolism and disposition in human lung cell models: comparison with in vivo expression profiles[END_REF]. B2B cells notably express active AhR and CYP1A1/1B1 proteins and secrete proinflammatory cytokines such as interleukin-(IL-) 6 and IL-8 [START_REF] Totlandsdal | Differential effects of the particle core and organic extract of diesel exhaust particles[END_REF]Le Vee et al., 2016). Different reports demonstrated that high concentrations of DEP can inhibit proliferation, induce DNA adducts and trigger IL-6 and IL-8 expression in B2B cells exposed for short time periods [START_REF] Pohjola | DNA binding of polycyclic aromatic hydrocarbons in a human bronchial epithelial cell line treated with diesel and gasoline particulate extracts and benzo[a]pyrene[END_REF]Cao et al., 2010;[START_REF] Totlandsdal | Differential effects of the particle core and organic extract of diesel exhaust particles[END_REF]. In addition, B2B cells exposed in vitro for four months to low concentrations of inorganic arsenic, a well-known carcinogenic metalloid, became tumorigenic once injected in nude mice through a mechanism involving ROS production and IL-6 overexpression [START_REF] Qi | Autophagy inhibition by sustained overproduction of IL6 contributes to arsenic carcinogenesis[END_REF]. Altogether, these results thus suggest that B2B cells could be an appropriate model to assess the longterm toxicity of environmental DEP concentrations.

The present study was designed to determine if chronic exposure to low DEP levels may alter the phenotype of B2B cells. Our results show that the repeated exposures of B2B cells for 6 months to 2 µg/ml (0.4 µg/cm 2 ) DEP (Standard Reference Material 1650b, National Institute of Standards and Technology) significantly increased the expression of CYP1A1 and 1B1 genes but induced neither stable modification of the B2B cell phenotype nor toxic effects related to DNA damage, oxidative stress or inflammation.

Materials and methods

Chemicals and reagents

DEP used in the present study was the Standard Reference Material 1650b (SRM 1650b), purchased from the National Institute of Standards and Technology (Gaithersburg,MD, USA). 4′,6-diamidino-2-phenylindole (DAPI) and primary antibody (Ab) directed against human phospho(ser139)-Histone H2AX (-H2AX) were purchased from Cell Signaling Technology (Ozyme, Montigny-le-Bretonneux, France). E-cadherin, N-cadherin and Vimentin Abs were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Mitomycine C (MMC) and H202 were purchased from Sigma-Aldrich (St-Quentin Fallavier, France). Tumor growth factor-β1 (TGF-β1) was obtained from Peprotech (Neuilly-Sur-Seine, France).

Cell culture

B2B cells were obtained from the American Type Culture Collection (CRL-9609). Cells were routinely cultured in complete Bronchial Epithelial Growth Medium (BEGM) (Lonza,Colmar, France) containing bronchial epithelial basal medium supplemented with gentamycin (50 μg/ml), hydrocortisone (0.5 μg/ml), retinoic acid (0.1 ng/ml), triiodotyronine (6.5 μg/ml), epinephrine (0.5 μg/ml), insulin (5 μg/ml), human EGF (0.5 ng/ml), transferrin (10 μg/ml) and bovine pituitary extract (52 μg/ml). Cells were cultured at 37°C in a humidified atmosphere with 5% CO2.

Preparation of DEP suspension and chronic exposure of B2B cells to DEP

A SRM 1650b unit consists of 200 mg diesel particulate matter generated by four-cycle diesel engines. This diesel particulate matter contains certified analytical concentrations of PAH (notably benzo(a)pyrene (B(a)P and phenanthrene) and nitro-PAH (notably 1-nitropyrene and 3-nitrophenanthrene), and induces DNA adducts in B2B cells exposed to high concentrations for 48 h [START_REF] Pohjola | DNA binding of polycyclic aromatic hydrocarbons in a human bronchial epithelial cell line treated with diesel and gasoline particulate extracts and benzo[a]pyrene[END_REF]. A stock suspension (5 mg/ml) was prepared by suspending DEP in sterile water containing 0.001% of triton-X100 and sonicating the suspension overnight at room temperature. According to the SRM1650b reference document, the mean particle diameter is 0.18 µm after a 24 h sonication.

To assess the chronic effects of DEP on B2B cell phenotype, cells were either not expose to DEP for 6 months to get the control passage-matched cell line B-CTR, or exposed to 2 µg/ml (0.4 µg/cm 2 ) DEP for six months to generate the B-DEP cell line. At day 1, cells were seeded at 10000 cells/cm 2 in a 25 cm 2 flask and cultured in complete BEGM in the absence or presence of DEP. At day 2 and day 5, the culture medium, with or without DEP, was renewed. At day 7, B-CTR and B-DEP cells were then detached using trypsin and seeded again in a 25 cm 2 flask. This procedure was reproduced weekly for both cell lines during the six months.

Then, different biological endpoints were compared in recently thawed B2B cells (passage 6 to 10), B-CTR cells and B-DEP cells, that were left untreated or exposed to DEP. To this goal, cells were then washed, detached using trypsin and seeded in the adequate culture plate. The next day, cells were not treated or treated with 2 µg/ml DEP for the indicated time intervals.

Soft agar assay

B-CTR, B-DEP and B2B cells were assessed for transformation by anchorage-independent growth in soft agar medium using the CytoSelect™ cell transformation assay, according to the manufacturer's protocol (Cell Biolabs, Clinisciences, Nanterre, France). Briefly, a suspension containing 10000 cells were transferred into each well of a 96-well flat-bottom microplate coated with a semisolid agar media and cultured for ten days in complete BEGM medium that did not contain DEP. At the end of the incubation, the cells were lysed after agar dissolution and quantified by the CyQuant® GR Dye using a fluorescence plate reader with excitation at 485 nm and emission detection at 530 nm. Each independent biological experiment was performed using three technical replicates and each result was the mean of the three replicates.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNAs were extracted by the TRIzol method and then analyzed by RT-PCR, in duplicate, using the fluorescent dye SYBR Green methodology and a CFX384 real-time PCR system (Bio-Rad, Marns-la-Coquette, France), as previously described (Macoch et al., 2015).

Predesigned primers for CYP1A1, CYP1B1, IL-6, and IL-8 gene expression analysis were provided by Sigma-Aldrich (St-Quentin Fallavier, France). Specificity of gene amplification was checked at the end of PCR through analysis of dissociation curves of the PCR products. Amplification curves were analyzed by the comparative cycle threshold method, using the CFX Manager™ Software. For each well, this software automatically sets the fluorescence baseline individually and then computes the optimal quantification cycle (Cq) on a per fluorophore basis by applying a multivariable, nonlinear regression model. Then, mean Cq values of two technical replicates were used to normalize expression of the steady-state target mRNA levels to those of the 18S ribosomal protein, using the 2(-ΔΔCq) method.

Western blot analysis

After treatments, cells were harvested and lysed for 20 min on ice in RIPA buffer supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1 mM orthovanadate, and a cocktail of protein inhibitors (Roche Diagnostic, Meylan, France). Cells were then centrifuged at 13000 g for 15 min at 4°C. The resulting supernatants were collected and frozen at 80°C or used immediately. Whole cell lysates were heated for 5 min at 95°C, loaded in a 4% stacking gel, and then separated by a 10% sodium dodecyl sulfate polymerase gel electrophoresis. Gels were electroblotted overnight onto nitro-cellulose membranes (Bio-Rad). After blocking with a Tris-buffered saline solution supplemented with 0.1% Tween20 and 5% bovine serum albumin, the membranes were hybridized with primary Abs overnight at 4°C and incubated with appropriate secondary horse-radish peroxidase-conjugated Abs.

Immunolabeled proteins were visualized by chemiluminescence. Densitometry analyses were carried out by normalizing levels of the target protein expression to those of p38-kinase expression.

Measurement of intracellular ROS levels

The intracellular ROS levels were determined using the dichlorodihydrofluoresceindiacetate (DCFH-DA) dye (Life Technology, Thermo Fischer Scientific, Courtaboeuf, France).

Cells were seeded at 50 000 cells/cm 2 in a 96-well culture plate and then incubated with 10 µM DCFH-DA for 30 min at 37°C. After incubation, cells were washed and DCF fluorescence intensity was measured using a Polar Star Omega fluorimeter (BMG LABTECH, Ozyme, Saint-Quentin-en-Yveline, France). H2O2 was used as a positive control to oxidize intracellular DCFH dye. Each independent biological experiment was performed using four technical replicates and each result was the mean of the four replicates.

Quantification of -H2AX histone foci by Immunofluorescence

Analysis of -H2AX foci was used to identify DNA double-strand breaks induced by DEP. Double-strand breaks are particularly hazardous as they can lead to genome rearrangements following deficient DNA repair and result in cell death or cancer.H2AX mediates cell cycle arrest and DNA repair following double-strands DNA breaks. It is activated by phosphorylation on its serine residue 139 (to form -H2AX) at site of DNA damage [START_REF] Kuo | Gamma-H2AX -a novel biomarker for DNA double-strand breaks[END_REF].

Briefly, B2B, B-CTR and B-DEP cells were seeded at 40 000 cells/cm 2 in 96-well culture plate.

After washing, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with PBS 0.2% triton-X100 for 10 min. Cells were washed three times with PBS, stained with primary Ab directed against -H2AX overnight at 4°C and then incubated with Alexa-647-conjugated IgG for 1 h. Nuclei were counterstained with DAPI and images were then acquired on an ArrayScan VTI high content screening reader with a 320 lens (Thermo Fischer Scientific, Courtaboeuf, France). The images were analyzed using the Cell Profiler software (http://www.cellprofiler.org, Broad Institute). In brief, spots corresponding to focal accumulations of -H2AX were detected in B2B, B-CTR, B-DEP cells treated or not with 2 µg/ml DEP for 24 h. For all analyses, the total amount of DAPI fluorescence and the total amount of Ab fluorescence per nucleus were indicated for each cell. MMC (100-500 µg/ml) was used as a positive control to induce DNA double-strand breaks and subsequent H2AX histone phosphorylation on its serine 139. Each independent biological experiment was performed using four technical replicates and each result was the mean of the four replicates.

Statistic analysis

Data were expressed as mean + standard deviation (SD) of the indicated number of independent biological experiments. Significant differences were evaluated using Student's t-test or ANOVA followed by the multi-range Dunett's t test when multiple comparisons were studied. Criterion of significance of the difference between means was p < 0.05.

Results

Determination of the relevant DEP concentration used for chronic B2B cell exposure

Many biological effects of DEP are mediated through activation of AhR and the subsequent induction of CYP1-metabolizing enzymes. Thus, to determine the concentration of DEP to be used to chronically expose B2B cells, we first determined the lowest concentration that could significantly increase CYP1A1 mRNA levels in B2B cells. To this purpose, cells were treated for 24 h with 0.1 to 5 µg/ml (0.02 to 1 µg/cm 2 ) DEP. As explained above, these concentrations constitute relevant environmental doses to which human bronchial epithelial cells may be exposed [START_REF] Li | Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects[END_REF]. Figure 1A shows that the 2 µg/ml DEP concentration was the lowest concentration increasing CYP1A1 mRNA levels. Maximal induction was measured in cells treated with 5 µg/ml DEP. We next determined if CYP1A1 mRNA levels were still induced when B2B cells were cultured for 6 days with 2 µg/ml DEP. As indicated in the Figure 1B, CYP1A1 mRNA levels were indeed significantly enhanced at day 6. Based on these data, we selected 2 µg/ml DEP to chronically expose B2B cells. Cells were treated for 6 months, as described in the Materials and Methods. Cells chronically treated with DEP were named B-DEP cells whereas their control passage-matched cells (24 to 28 passages) were named B-CTR cells.

In addition, B2B cells refer to cells that were cultured in the absence of DEP for a maximum of 6 to 10 passages.

Basal and DEP-induced CYP1 mRNA levels are similar in B2B, B-CTR and B-DEP cells

In the next steps of this study, we investigated stable modifications of the phenotype of cells chronically exposed to 2 µg/ml DEP. In order to avoid characterization of the effects induced by the latest DEP treatments performed during chronic exposure, cells were washed three times, detached and seeded in new culture plates. The next day, B2B, B-CTR and B-DEP cells were left untreated or treated for 6 days with 2 µg/ml DEP. After 6 days, basal CYP1A1 and CYP1B1 mRNA levels were similar in the three cell lines (Figure 2A). Figure 2B clearly shows that CYP1A1/1B1 mRNA levels were significantly increased, with a similar intensity, in B2B, B-CTR and B-DEP cells treated for 6 days with 2 µg/ml DEP (Figure 2B). Thus, chronic DEP exposure did not impact the ability of DEP to induce CYP1 gene expression in B2B cells.

Chronic DEP exposure does not modify the phenotype of B2B cells

We next investigated if long term culture in the presence of DEP could affect cell morphology, induce epithelial-mesenchymal transition (EMT) and/or stimulate anchorageindependent cell growth. First, the Figure 3A shows that chronic DEP exposure did not modify the morphology of B2B cells that remained cuboid. We assessed the ability of DEP to induce EMT or to promote TGF-1-induced EMT by measuring protein levels of typical epithelial (Ecadherin) and mesenchymal (N-cadherin and vimentin) biomarkers. Cells were washed, detached and seeded in new culture plates. The next day, cells were left untreated or stimulated with 10 ng/ml TGF-1, in the absence of DEP, for four days. Figure 3B shows that basal E-cadherin, N-cadherin and vimentin protein levels remained similar in B2B, B-CTR and B-DEP cells. As expected, TGF-1 stimulation strongly repressed E-cadherin expression and significantly induced those of N-cadherin and vimentin. Chronic exposure of B2B cells to DEP neither prevented nor increased EMT induced by TGF-1. It can be noted however that, although TGF-1 significantly increased N-cadherin and vimentin expressions in B-CTR cells, it only weakly repressed E-cadherin expression. This suggests that long term culture of B2B cells, in the absence of DEP, could have modified TGF-1 signaling. B2B cells are not tumorigenic in immunocompromised mice but did form colonies in semisolid agar medium [START_REF] Qi | Autophagy inhibition by sustained overproduction of IL6 contributes to arsenic carcinogenesis[END_REF]. Indeed, we found that B2B cells markedly proliferated when cultured for 10 days in agar semisolid medium. However, at day 10, the numbers of cells measured for the three cell lines remained similar, which suggests that chronic DEP exposure did not amplify the anchorage-independent growth of B2B cells (Figure 3C).

Impact of chronic DEP exposure on ROS production, -H2AX levels and pro-inflammatory gene expression

Because ROS production, genotoxicity and inflammation are potent molecular mechanisms by which DEP could exert their toxicity, we next analyzed different endpoints associated with these mechanisms of action in B2B, B-CTR and B-DEP cells.

ROS levels were analyzed by quantifying DCF fluorescence in cells seeded in new culture plates and, the next day, left untreated or treated with 2 µg/ml DEP for 24 h. Basal ROS production was identical in B2B, B-CTR and B-DEP cells and was not increased in any cell lines treated with 2 µg/ml DEP (Figure 4A). Cell treatment, with 2 µg/ml DEP for only 1 h or 6 h, did not modified ROS levels as well (data not shown). In contrast, basal fluorescence intensity measured in B2B cells (1615 + 63.3 relative fluorescence units) was markedly increased by 1 h stimulation with H2O2, used as a positive control, and reached 58568 + 14931 relative fluorescence unit, which confirm the sensitivity of B2B cells to ROS. In addition, we measured mRNA levels of NADPH quinone oxidoreductase 1 (NQO1) gene which are generally increased in response to oxidative cell stress and/or ROS production. Figure 4B shows that NQO1 mRNA levels were similar in B2B, B-CTR and B-DEP cells and, thus, not modified by the chronic DEP exposure. We next assessed the ability of DEP to induce DNA double-strand breaks by measuring the intensity of nuclear foci of -H2AX. Finally, we determined if DEP could up-regulate expression of pro-inflammatory genes in chronically exposed B2B cells. To this goal, we compared in the three cell lines mRNA levels of IL-6 and IL-8 genes. Cells were seeded in new plates, and the next day, cells were cultured for 24 h in the absence or presence of 2 µg/ml DEP. Basal IL-6 and IL-8 mRNA levels were similar in B2B, B-CTR and B-DEP cell lines (Figure 4C). DEP treatment for 24 h did not modify IL-6 and IL-8 gene expression in any cell lines (data not shown).

In the present study, we show that chronic exposure of the human bronchial epithelial B2B cells to low environmental concentrations of DEP significantly increased CYP1 gene expressions but did not induce cancer-related modifications of the cell phenotype.

First, our results demonstrate that B-DEP cells, exposed for 6 months to 2 µg/ml DEP, control matched-passage B-CTR cells, and parental B2B cells displayed similar basal and DEP-induced levels of CYP1A1 and CYP1B mRNA. Thus, the chronic exposure to DEP neither permanently increased CYP1 gene expression nor alter the sensitivity of B-DEP cells to a new DEP treatment. Two µg/ml (0.4 µg/cm 2 ) represents the lowest concentration of DEP inducing CYP1A1 expression in B2B cells treated for 24 h. Totlansdal et al. (2015) reported that DEP, generated from a diesel engine different from that producing the SRM 1650b DEP, could significantly increase CYP1A1 mRNA levels in B2B cells, when used at a concentration as low as 0.013 µg/cm 2 . These results indicate that CYP1A1 gene expression is thus a very sensitive endpoint that can be measured to assess the effects of environmental DEP concentrations in B2B cells. However, the expression of CYP1A1 may not constitute a general cell biomarker highlighting DEP exposure since CYP1A1 expression is also induced by many other chemicals.

CYP1A1 gene is specifically induced in response to AhR activation and its expression is probably controlled by AhR agonists desorbed from DEP. In this context, the referent document indicates that SRM 1650b DEP contain several PAH and nitro-PAH such as B(a)P, benzo(a)anthracene, chrysene and nitro-pyrene which are potent AhR agonists. CYP1A1 and 1B1 metabolize these PAH into electrophilic metabolites that mediate their carcinogenic effects. For example, the metabolite of B(a)P, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE), is cytotoxic, mutagenic and carcinogenic [START_REF] Moorthy | Polycyclic aromatic hydrocarbons: from metabolism to lung cancer[END_REF]. In B2B cells, this metabolite induces stable expression of cyclin D1 by a mechanism involving a phosphatidylinositol 3kinase/AKT/MAPK pathway. Up-regulation of cyclin D1 plays a major role in oncogenic transformation and tumorigenesis of B2B cells treated with 0.5 µM BPDE for 12 weeks [START_REF] Ding | Cyclin D1 induction by benzo[a]pyrene-7,8-diol-9,10-epoxide via the phosphatidylinositol 3-kinase/Akt/MAPK-and p70s6k-dependent pathway promotes cell transformation and tumorigenesis[END_REF]. In addition, chronic treatment of B2B with 1 µM BPDE downregulates expression of the PHLPP2 phosphatase that leads to sustained Tumor Necrosis Factor  (TNF-) gene induction. Besides cyclin D1, up-regulation of TNF- is critical for B(a)P/BPDE-induced anchorage-independent growth in soft agar and tumorigenesis in mice [START_REF] Huang | PHLPP2 Downregulation Contributes to Lung Carcinogenesis Following B[a]P/B[a]PDE Exposure[END_REF].

Finally, B(a)P induces the formation of BPDE-DNA adducts and significantly increases the number of micronuclei in B2B cells [START_REF] Van Agen | B[a]P-DNA adduct formation and induction of human epithelial lung cell transformation[END_REF].

Our results show that chronic exposure to 2 µg/ml DEP (SRM 1650b) did not stimulate the ability of B2B cells to proliferate in soft agar medium or increase the formation of -H2AX foci.

The amounts of PAH or nitro-PAH desorbed from DEP were sufficient to induce CYP1A1 gene expression but were likely too low to generate concentrations of ultimate carcinogenic metabolites able to promote cell transformation or exert mutagenic effects in B2B cells. This idea is supported by the results published by [START_REF] Pohjola | DNA binding of polycyclic aromatic hydrocarbons in a human bronchial epithelial cell line treated with diesel and gasoline particulate extracts and benzo[a]pyrene[END_REF] which demonstrated that 15 µg/ml SRM 1650 DEP only slightly increased DNA adducts in B2B cells exposed for 48 h.

Similarly, our study indicates that the repeated treatments of B2B cells with 2 µg/ml DEP did not modify ROS levels or IL-6/IL-8 expression. Totlandsdal et al. (2003) reported that 25 to 100 fold higher concentrations (50-200 µg/ml) of DEP were necessary to stimulate IL-6 and IL-8 release from B2B cells exposed for 24 h. It is also noteworthy that DEP did not trigger EMT, a major developmental process that favors migration/invasion and metastasis of epithelial cancer cells [START_REF] Roche | Epigenetic Regulation of the Epithelial to Mesenchymal Transition in Lung Cancer[END_REF]. EMT is nevertheless induced in B2B cells chronically exposed to total particulate matter from cigarette smoke that is known to contain carcinogenic PAH such as B(a)P (van der [START_REF] Van Der Toorn | The biological effects of long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product[END_REF].

In contrast to DEP, different reports have clearly demonstrated that long term exposure to low concentrations of inorganic arsenic, in the range of plasmatic arsenic levels measured in chronically exposed individuals, promoted tumorigenic transformation of B2B cells by different molecular mechanisms involving sustained ROS levels, up-regulated IL-6 expression and EMT [START_REF] Zhang | Autophagy is a cell self-protective mechanism against arsenic-induced cell transformation[END_REF][START_REF] Qi | Autophagy inhibition by sustained overproduction of IL6 contributes to arsenic carcinogenesis[END_REF][START_REF] Yang | Inactivation of miR-100 combined with arsenic treatment enhances the malignant transformation of BEAS-2B cells via stimulating epithelial -mesenchymal transition[END_REF]. B2B cells may thus constitute an adequate in vitro model to investigate molecular mechanisms of some, but not all, carcinogenic chemicals. Particularly, our results show that low environmentally relevant concentrations of DEP did not induce cancer-related effects in chronically exposed B2B cells. Moreover, it should be noted that an in vivo experimental study has recently reported that chronic exposure of rats to a high, but realistic, concentration of DEE for periods of 3 h every day, for 5 days, during 3 weeks, did not modified ROS levels, -H2AX foci or induce DNA adducts in lungs [START_REF] Douki | Comparative study of diesel and biodiesel exhausts on lung oxidative stress and genotoxicity in rats[END_REF]. Moreover, epidemiological studies have shown that the increased risk of human lung cancer associated with occupational exposure to diesel emission is only modest [START_REF] Möhner | A critical review of the relationship between occupational exposure to diesel emissions and lung cancer risk[END_REF]. Thus, whatever the cell model used, it must be difficult to highlight the in vitro carcinogenic effects of low environmental concentrations of DEP.

In conclusion, our study demonstrates that DEP similarly increases CYP1A1 expression in the human bronchial epithelial B2B (BEAS-2B) cell line unexposed or exposed for 6 months to environmentally relevant concentrations of DEPs. Moreover, we show that chronic exposure to these low DEP concentrations induces neither molecular mechanism nor phenotype modification related to genotoxic, pro-oxidant or inflammatory effects of DEP. Altogether, these results strongly suggest that the long-term toxicity of the SRM 1650b DEP is thus very limited in this cell line. In the future, chronic DEP effects towards human lung cells should be further addressed by assessing in vitro chronic toxicity of DEP collected from more recent engines, using classical 2D and/or newly developed 3D cell models.

Acknowledgments

This work was funded by the Institut national de la santé et de la recherche médicale (INSERM) and by the Université de Rennes (Univ Rennes). We are grateful to Remy Le Guevel from the ImPACcell platform (SFR UMS CNRS 3480 -INSERM 018, Biosit, Univ Rennes, Rennes, France) for image acquisition and quantification of -H2AX foci.

Cao, D., Bromberg, P.A., Samet, J.M., 2010. Diesel particle-induced transcriptional expression of p21 involves activation of EGFR, Src, and Stat3. Am. J. Respir. Cell Mol. Biol. 42, 88-95.

Legends

Figure 1: Induction of CYP1A1 mRNA expression by DEP in B2B cells. Cells were exposed to 0.1 to 5 µg/ml DEP for 24 h (A) and to 2 µg/ml DEP for 6 days (B). mRNA levels of CYP1A1 were determined by quantitative RT-PCR and normalized to endogenous ribosomal 18S RNA levels. Data are expressed relatively to mRNA levels found in "0" untreated cells, arbitrarily set at the value of 1. All results are expressed as means + SD of four independent biological experiments. *p < 0.05, ***p < 0.001 versus "0".

  Figure 4C (left panel) clearly demonstrates that MMC, used as a positive control, strongly and significantly increased the formation of -H2AX foci in B2B cells treated for 24 h. However, -H2AX foci remained identical in B-CTR and B-DEP cells indicating that the chronic DEP treatment did not induce DNA double-strand breaks (Figure 4C, right panel).

Figure 2 :

 2 Figure 2: Basal and DEP-induced CYP1A1/CYP1B1 mRNA levels in B2B, B-DEP and B-

Figure 3 :

 3 Figure 3: Morphology and phenotypes of B2B, B-CTR and B-DEP cells. (A) Confluent

Figure 4 :

 4 Figure 4: ROS, -H2AX levels and pro-inflammatory gene expression in B2B, B-CTR and

  Figure1


Mary Gemmel

Ph.D Jodi L Pawluski
email: j.pawluski@gmail.com

Perinatal SSRI medications and offspring hippocampal plasticity: interaction with maternal stress and sex Abbreviated title: Perinatal SSRIs and hippocampus

Keywords: Antidepressants, Perinatal Depression, Prenatal Stress, Neurogenesis, Hippocampus, Adolescence, Sex Differences, 5-HT, serotonin

Perinatal SSRI medications and offspring hippocampal

plasticity interaction with maternal stress and sex

Introduction

Up to 20% of women are diagnosed with depression or anxiety during the perinatal period. [START_REF] Almond | Postnatal depression: a global public health perspective[END_REF][START_REF] Burke | Depression and cortisol responses to psychological stress: a meta-analysis[END_REF][START_REF] Leung | Perinatal depression: prevalence, risks, and the nutrition link--a review of the literature[END_REF][START_REF] Pawluski | The Neurobiology of Postpartum Anxiety and Depression[END_REF] These affective disorders can have detrimental effects for both the mother and developing child and as such, treatment is needed. [START_REF] Pawluski | The Neurobiology of Postpartum Anxiety and Depression[END_REF][START_REF] Oberlander | Disentangling Maternal Depression and Antidepressant Use During Pregnancy as Risks for Autism in Children[END_REF] Many antidepressant treatments are available to treat maternal affective disorders, with selective serotonin reuptake inhibitor medications (SSRIs) being the recommended first line of treatment. [START_REF] Pawluski | The Neurobiology of Postpartum Anxiety and Depression[END_REF][START_REF] Oberlander | Neonatal outcomes after prenatal exposure to selective serotonin reuptake inhibitor antidepressants and maternal depression using population-based linked health data[END_REF][START_REF] Cooper | Increasing use of antidepressants in pregnancy[END_REF] SSRIs, with the exception of paroxetine, are considered safe for use during pregnancy as they have no major teratogenic effects on the fetus. [START_REF] Gentile | The safety of newer antidepressants in pregnancy and breastfeeding[END_REF] SSRI prescription rates for pregnant women in industrialized countries (including Canada, Iceland, Denmark, Sweden, UK, Italy, the Netherlands, and France) range between 2-7% in Canada and Western European countries, and between 5-13% in Australia and the USA. [START_REF] Oberlander | Neonatal outcomes after prenatal exposure to selective serotonin reuptake inhibitor antidepressants and maternal depression using population-based linked health data[END_REF][START_REF] Cooper | Increasing use of antidepressants in pregnancy[END_REF][START_REF] Hayes | Maternal antidepressant use and adverse outcomes: a cohort study of 228,876 pregnancies[END_REF][START_REF] Charlton | Selective serotonin reuptake inhibitor prescribing before, during and after pregnancy: a population-based study in six European regions[END_REF][START_REF] Zoega | Use of SSRI and SNRI Antidepressants during Pregnancy: A Population-Based Study from Denmark, Iceland, Norway and Sweden[END_REF][START_REF] Lupattelli | Medication use in pregnancy: a cross-sectional, multinational web-based study[END_REF] These medications are expected to promote maternal mental health and thus promote the health and wellbeing of both the mother and child. [START_REF] Kim | Pharmacotherapy of postpartum depression: an update[END_REF] However, SSRIs can cross the placenta and, to a lesser degree, are found in breast milk, modulating the serotonergic environment for the developing fetus and having potentially long-term effects on neurodevelopment. [START_REF] Oberlander | Sustained neurobehavioral effects of exposure to SSRI antidepressants during development: molecular to clinical evidence[END_REF][START_REF] Homberg | New perspectives on the neurodevelopmental effects of SSRIs[END_REF][START_REF] Glover | Of rodents and humans: A comparative review of the neurobehavioral effects of early life SSRI exposure in preclinical and clinical research[END_REF][START_REF] Gemmel | Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: Interactions with pre-gestational maternal stress[END_REF] Clinical research has shown that children exposed to SSRIs prenatally are at increased risk for behavioral abnormalities including attentional deficits, neuropsychiatric disorders, and neurodevelopmental disorders. [START_REF] Clements | Prenatal antidepressant exposure is associated with risk for attention-deficit hyperactivity disorder but not autism spectrum disorder in a large health system[END_REF][START_REF] Man | Exposure to selective serotonin reuptake inhibitors during pregnancy and risk of autism spectrum disorder in children: a systematic review and meta-analysis of observational studies[END_REF][START_REF] Oberlander | Externalizing and attentional behaviors in children of depressed mothers treated with a selective serotonin reuptake inhibitor antidepressant during pregnancy[END_REF] As the hippocampus is a key mediator of behavior and cognition, and receives substantial serotoninergic innervation, such effects are likely linked to SSRI-related changes in hippocampal development. Therefore, this mini review will focus on how perinatal SSRIs affect plasticity in the developing hippocampus. First, we will briefly review how SSRIs during the perinatal period may alter the developing serotoninergic system, and then we will review what is known about perinatal SSRI effects on biomarkers of neuroplasticity in clinical studies and measures of hippocampus plasticity from work in laboratory rodent models. Early life exposure to maternal mental illness, in the presence of SSRIs, can also have a persistent effects of offspring neurobehavioral outcomes. [START_REF] Brown | Association Between Serotonergic Antidepressant Use During Pregnancy and Autism Spectrum Disorder in Children[END_REF][START_REF] Mezzacappa | Risk for Autism Spectrum Disorders According to Period of Prenatal Antidepressant Exposure: A Systematic Review and Meta-analysis[END_REF] Thus, the implications of both perinatal SSRIs and maternal stress-related disorders will be discussed in terms of offspring outcomes, where possible. [START_REF] Haim | Gestational stress induces persistent depressive-like behavior and structural modifications within the postpartum nucleus accumbens[END_REF][START_REF] O'mahony | Gestational stress leads to depressive-like behavioural and immunological changes in the rat[END_REF] Understanding the long-term effect of perinatal SSRIs on hippocampus plasticity will aid in understanding the risks and benefits of exposure to these medications during the perinatal period.

Serotonin, SSRIs and the developing hippocampus

During development, serotonin (5-hydroxytryptamine or 5-HT) plays an important role in regulating proliferation, migration, and differentiation of neurons as well as axonal connectivity and synaptic pruning. [START_REF] Gaspar | The developmental role of serotonin: news from mouse molecular genetics[END_REF][START_REF] Herlenius | Development of neurotransmitter systems during critical periods[END_REF][START_REF] Pm | Serotonin and brain development: role in human developmental diseases[END_REF] A number of studies indicate that serotonin signaling during early life is critically involved in the development of neuronal circuits. [START_REF] Pm | Serotonin and brain development: role in human developmental diseases[END_REF][START_REF] Ansorge | Inhibition of serotonin but not norepinephrine transport during development produces delayed, persistent perturbations of emotional behaviors in mice[END_REF][START_REF] Ansorge | Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice[END_REF][START_REF] Fernandez | Constitutive and Acquired Serotonin Deficiency Alters Memory and Hippocampal Synaptic Plasticity[END_REF][START_REF] Kurzepa | The 5HT level and MAO activity in rat brain during development[END_REF][START_REF] Baker | 5-hydroxytryptamine metabolism in early embryogenesis, and the development of brain and retinal tissues. A review[END_REF] Therefore, any changes to serotonin levels and/or serotonergic system functioning during the perinatal period could disrupt the development of not only the serotonergic system in infants, but also countless brain regions which receive serotonergic input, such as the hippocampus.

SSRIs act by blocking the serotonin transporter (SERT) on the presynaptic neuronal membrane and subsequently reduce serotonin reuptake, increasing serotonin's concentration in the synaptic cleft, and aiding in chemical transmission between neurons which can have moodstabilizing effects. As perinatal SSRIs target maternal SERT activity, and are present in both the placenta and at much lower levels in breast milk, [START_REF] Kristensen | Distribution and excretion of fluoxetine and norfluoxetine in human milk[END_REF] the use of such medications during pregnancy and lactation suggest an effect on infant neural development. The consequences of perinatal SSRI exposure may not occur immediately after birth, but may manifest later during childhood, adolescence or even in adulthood. [START_REF] Oberlander | Sustained neurobehavioral effects of exposure to SSRI antidepressants during development: molecular to clinical evidence[END_REF][START_REF] Gemmel | Perinatal selective serotonin reuptake inhibitor medication (SSRI) effects on social behaviors, neurodevelopment and the epigenome[END_REF][START_REF] Pawluski | Perinatal selective serotonin reuptake inhibitor exposure: impact on brain development and neural plasticity[END_REF] Perinatal SSRI treatment can increase hippocampal serotonin levels in adolescent and adult male mice [START_REF] Ishiwata | Selective serotonin reuptake inhibitor treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction[END_REF] and pre-adolescent male and female offspring. [START_REF] Gemmel | under review Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: interactions with maternal stress[END_REF] Interestingly, early life exposure to SSRIs also normalizes hippocampal levels of serotonin and serotonin's metabolite 5hydroxyindolacetic acid (5-HIAA) in prenatally restraint stressed offspring at weaning, [START_REF] Gemmel | Developmental fluoxetine and prenatal stress effects on serotonin, dopamine, and synaptophysin density in the PFC and hippocampus of offspring at weaning[END_REF] serotonergic transmission in the ventral hippocampus of adolescent rat offspring after prenatal dexamethasone treatment, [START_REF] Nagano | Early intervention with fluoxetine reverses abnormalities in the serotonergic system and behavior of rats exposed prenatally to dexamethasone[END_REF] and serotonin turnover (via 5-HIAA/5-HT ratios) in the hippocampus of prenatally stressed male mouse offspring (Table 1). [START_REF] Ishiwata | Selective serotonin reuptake inhibitor treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction[END_REF] Although not well studied in the hippocampus, perinatal SSRIs do affect serotonergic receptors in cortical and hypothalamic brain regions and thus many layers of the serotonergic system are likely altered with perinatal SSRIs. 40- 42 It should not be forgotten that maternal stress can also have enduring effects on serotonin, even when combined with perinatal SSRIs: Recently we have shown that pre-gestational chronic unpredictable stress results in significantly reduced hippocampal serotonin levels in pre-adolescent offspring, particularly in female offspring, regardless of perinatal fluoxetine exposure. [START_REF] Gemmel | Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: Interactions with pre-gestational maternal stress[END_REF][START_REF] Gemmel | Perinatal selective serotonin reuptake inhibitor medication (SSRI) effects on social behaviors, neurodevelopment and the epigenome[END_REF] Others have shown that pre-gestational stress increases serotonin levels and decreases serotonin transporter expression in the hippocampus of fetal rats, [START_REF] Huang | Pre-gestational stress reduces the ratio of 5-HIAA to 5-HT and the expression of 5-HT1A receptor and serotonin transporter in the brain of foetal rat[END_REF] and that prenatal stress increases serotonin in weanling offspring, regardless of fluoxetine exposure. Changes in early-life serotonin, and serotonin transporters, may have enduring effects on serotonergic functioning of offspring later in life. Together these findings show that there are long-term effects of both SSRIs and maternal stress on the serotonin system of the hippocampus and that the timing and duration of maternal stress (pre-gestational versus prenatal), as well as offspring age and sex, may mediate these effects on the developing serotonergic system. Therefore, understanding how fluctuations in serotonin and the serotonergic system impact developing neuroplasticity via early life SSRIs, and maternal stress, is critical in integrating what we know about hippocampal plasticity in offspring.

Perinatal SSRIs and biomarkers of neuroplasticity in Clinical Research

Findings from clinical studies start to point to an effect of prenatal SSRIs on fetal and neonatal neurodevelopment by investigating biomarkers in peripheral fluids related to central nervous system development. It has been reported that prenatal SSRIs decreases S100B, an astroglial-specific Ca2+-binding protein, [START_REF] Haring | Hippocampal serotonin levels influence the expression of S100 beta detected by immunocytochemistry[END_REF][START_REF]Stimulation of astroglial 5-HT1A receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology[END_REF] in human neonates at birth. [START_REF] Pawluski | Neonatal S100B protein levels after prenatal exposure to selective serotonin reuptake inhibitors[END_REF] S100B mediates the positive outgrowth and survival of neurons, [START_REF] Bhattacharyya | S100 is present in developing chicken neurons and Schwann cells and promotes motor neuron survival in vivo[END_REF][START_REF] Gonzalez-Martinez | S-100 proteins in the human peripheral nervous system[END_REF] and stimulates glial cell proliferation [START_REF] Selinfreund | Neurotrophic protein S100 beta stimulates glial cell proliferation[END_REF] ; therefore, S100B levels in human biological fluids may be a useful indicator of brain maturation and suggest an impact of prenatal drug exposure on neural development. [START_REF] Pawluski | Neonatal S100B protein levels after prenatal exposure to selective serotonin reuptake inhibitors[END_REF] Since this reduction in S100B occurs when controlling for maternal levels of depression, which serves as a confounding variable in clinical work [START_REF] Oberlander | Disentangling Maternal Depression and Antidepressant Use During Pregnancy as Risks for Autism in Children[END_REF] , such outcomes suggest a significant effect of perinatal SSRI exposure itself on potential markers of neurodevelopment. In addition, reelin levels are decreased in neonates prenatally exposed to SSRIs. [START_REF] Brummelte | Antidepressant use during pregnancy and serotonin transporter genotype (SLC6A4) affect newborn serum reelin levels[END_REF] Reelin is an important glycoprotein which plays a critical role in neuronal migration and positioning during neurodevelopment, and these findings further suggest an effect of early life changes in serotonin, via SSRIs, on the brain of developing offspring. Recent imaging data has confirmed that prenatal SSRIs do indeed alter the developing brain, at least in very preterm infants, with prenatal SSRI exposed infants showing decreased activity in the basal ganglia and thalamus. [START_REF] Podrebarac | Antenatal exposure to antidepressants is associated with altered brain development in very preterm-born neonates[END_REF] However, due to limitations in the ability of clinical research to investigate central neurodevelopment in SSRI-exposed offspring, animal models have been used to gain insight into the specific effects of perinatal SSRIs on neurogenesis and synaptic plasticity in the hippocampus. Below is a summary of findings on how perinatal SSRIs affect the developing hippocampus of male and female offspring.

Perinatal SSRIs and plasticity in the hippocampus

With clinical work pointing to an effect of perinatal SSRIs on both S100B and reelin, key players in neural migration and plasticity, it is not surprising that there is growing interest in the long-term impact of SSRIs on hippocampal neurogenesis and plasticity. The hippocampus is one of two brain regions where there is a remarkably high rate of neurogenesis throughout the lifespan (the other region being the subventricular zone). [START_REF] Eriksson | Neurogenesis in the adult human hippocampus[END_REF][START_REF] Pawluski | Effects of steroid hormones on neurogenesis in the hippocampus of the adult female rodent during the estrous cycle, pregnancy, lactation and aging[END_REF] These new neurons, and their ability to form and establish new connections, in the hippocampus play an important role in learning and memory, stress regulation, and, more recently, social behaviours. [START_REF] Pawluski | Effects of steroid hormones on neurogenesis in the hippocampus of the adult female rodent during the estrous cycle, pregnancy, lactation and aging[END_REF][START_REF] Hammels | Differential susceptibility to chronic social defeat stress relates to the number of Dnmt3a-immunoreactive neurons in the hippocampal dentate gyrus[END_REF][START_REF] Dalla | Stressful experience has opposite effects on dendritic spines in the hippocampus of cycling versus masculinized females[END_REF][START_REF] Leuner | Structural plasticity and hippocampal function[END_REF][START_REF] Djavadian | Serotonin and neurogenesis in the hippocampal dentate gyrus of adult mammals[END_REF] The hippocampus consists of three main regions: cornu ammonis 1 (CA1), cornu ammonis 3 (CA3), and the dentate gyrus, the lattermost being a site with a high rate of continuous neurogenesis in adulthood (Figure 1). [START_REF] Altman | Are new neurons formed in the brains of adult mammals?[END_REF][START_REF] Altman | Postnatal neurogenesis in the guinea-pig[END_REF] Understanding how neurogenesis and neuroplasticity may shift following perinatal SSRIs will further clarify the role that early-life exposures have on neurobehavioral development.

To date, findings highlight long-term effects of perinatal SSRIs on hippocampal plasticity in rodent offspring, particularly after maternal stress. These studies have primarily focused on measures of brain derived neurotrophic factor (BDNF), neurogenesis, and synaptic modifications in the hippocampus. Of particular importance are alterations of BDNF signaling, epigenetic changes at the BDNF gene, and changes in hippocampal plasticity, which have been extensively implicated in the pathophysiology and treatment of mood disorders during adulthood. [START_REF] Boulle | Epigenetic regulation of the BDNF gene: implications for psychiatric disorders[END_REF][START_REF] Castren | The role of BDNF and its receptors in depression and antidepressant drug action: Reactivation of developmental plasticity[END_REF] Developmental exposure to SSRIs has resulted in long-term impact on BDNF in offspring; Early developmental treatment with fluoxetine can induce long-lasting behavioral impairment, via increased inhibition to stressful events and reduced behavioral despair, accompanied with upregulation of hippocampal BDNF mRNA and TrkB mRNA levels in adult male mice [START_REF] Karpova | Long-lasting behavioural and molecular alterations induced by early postnatal fluoxetine exposure are restored by chronic fluoxetine treatment in adult mice[END_REF] and a decrease in global DNA methylation in the hippocampus of male rats [START_REF] Toffoli | Maternal exposure to fluoxetine during gestation and lactation affects the DNA methylation programming of rat's offspring: modulation by folic acid supplementation[END_REF] . Serotonin transporter (5-HTT) knockout rats, which have elevated levels of serotonin throughout life, display decreased BDNF levels in the hippocampus, concomitant with increased DNA methylation at BDNF promoters IV and VI. [START_REF] Molteni | Reduced function of the serotonin transporter is associated with decreased expression of BDNF in rodents as well as in humans[END_REF] A exact comparison between perinatal SSRI exposure and 5-HTT KO rat models with regards to BDNF levels and methylation status has not been carried, however it is likely that the apparent differences between models on BDNF effects in the hippocampus are due to the fact that 5-HTT KO animals have elevated levels of serotonin throughout life and not just during the perinatal period. While this work suggests an effect of perinatal SSRI exposure on BDNF, these three previous studies did not investigate female offspring. We have recently reported in adult female rat offspring that early life exposure to SSRIs (fluoxetine) increases immobility in the forced swim test, decreases hippocampal BDNF exon IV mRNA levels, and increases levels of the repressive histone 3 lysine 27 tri-methylated mark at the corresponding promoter. [START_REF] Boulle | Developmental fluoxetine exposure increases behavioral despair and alters epigenetic regulation of the hippocampal BDNF gene in adult female offspring[END_REF] We also found a significant negative correlation between hippocampal BDNF exon IV mRNA levels and immobility in the forced swim test with higher hippocampal BDNF mRNA expression being associated with less immobility in the forced swim test. [START_REF] Boulle | Developmental fluoxetine exposure increases behavioral despair and alters epigenetic regulation of the hippocampal BDNF gene in adult female offspring[END_REF] In adult male offspring, developmental fluoxetine exposure decreased BDNF IV and TrkB mRNA expression in the hippocampus and these changes were not associated with changes in the immobility measure. [START_REF] Boulle | Prenatal stress and early-life exposure to fluoxetine have enduring effects on anxiety and hippocampal BDNF gene expression in adult male offspring[END_REF] In addition, adult male offspring showed an enduring effect of prenatal stress in decreasing hippocampal BDNF IV mRNA expression, [START_REF] Boulle | Prenatal stress and early-life exposure to fluoxetine have enduring effects on anxiety and hippocampal BDNF gene expression in adult male offspring[END_REF] further demonstrating these effects sexually differentiated effects following SSRI exposure. Regardless, perinatal SSRIs effects on epigenetic modifications and changes to BDNF signaling, a critical growth factor required for neuronal growth and development, suggest an impact on additional aspects of neurodevelopment such as neurogenesis and neuroplasticity.

Early life exposure to SSRIs reduces cell proliferation in the hippocampus of adolescent offspring. [START_REF] Rayen | Fluoxetine during development reverses the effects of prenatal stress on depressive-like behavior and hippocampal neurogenesis in adolescence[END_REF] However, when combined with a model of maternal stress, early life exposure to SSRIs prevents the effects of maternal stress on hippocampal neurogenesis (immature neurons), in adolescence and adulthood. [START_REF] Rayen | Fluoxetine during development reverses the effects of prenatal stress on depressive-like behavior and hippocampal neurogenesis in adolescence[END_REF][START_REF] Rayen | Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-dependent effects on hippocampal plasticity[END_REF] These effects occur in both male and female offspring. Others have shown similar effects with postnatal SSRIs preventing the reduction in hippocampal cell proliferation and increased cell death observed in juvenile rat offspring subjected to premature maternal separation. [START_REF] Lee | Fluoxetine enhances cell proliferation and prevents apoptosis in dentate gyrus of maternally separated rats[END_REF] Developmental exposure to fluoxetine can also have a sexually differentiated effect on the number of immature neurons after maternal corticosterone treatment, [START_REF] Gobinath | Maternal postpartum corticosterone and fluoxetine differentially affect adult male and female offspring on anxiety-like behavior, stress reactivity, and hippocampal neurogenesis[END_REF] with maternal postpartum fluoxetine increasing the density of immature neurons via doublecortin expression in the hippocampus of adult male offspring, but decreasing the density of immature neurons in adult female offspring. Interestingly, additional reports found no effects of perinatal fluoxetine on hippocampal neurogenesis in the dorsal hippocampus at weaning or in preadolescent offspring. [START_REF] Gemmel | under review Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: interactions with maternal stress[END_REF][START_REF] Gemmel | Developmental fluoxetine and prenatal stress effects on serotonin, dopamine, and synaptophysin density in the PFC and hippocampus of offspring at weaning[END_REF] Thus, it appears that after adolescence and puberty, the effects of perinatal SSRIs on hippocampal neurogenesis and plasticity become pronounced and sexually differentiated.

With regards to perinatal SSRI effects on spine densities and synaptic proteins, these effects appear to be primarily dependent on offspring age and sex. For example, early postnatal treatment with fluoxetine reverses the reduction in CA3 spine and synapse density observed in prenatally stressed juvenile and adolescent male mice (females were not studied). [START_REF] Ishiwata | Selective serotonin reuptake inhibitor treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction[END_REF] However, in rat offspring at weaning, we found no effect of early life exposure to fluxotine on pre-synaptic protein densities (via synaptophysin-immunoreactivity) in the CA3 or dentate gyrus. [START_REF] Gemmel | Developmental fluoxetine and prenatal stress effects on serotonin, dopamine, and synaptophysin density in the PFC and hippocampus of offspring at weaning[END_REF] Effects on spine density appear to emerge during pre-adolescence, with pre-adolescent female, not male, rat offspring perinatally exposed to fluoxetine showing increased hippocampal pre-synaptic density in the dentate gyrus. [START_REF] Gemmel | under review Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: interactions with maternal stress[END_REF] These effects are region-specific, with perinatal fluoxetine exposure increasing CA2 density in pre-adolescent females, but decreasing such density in combination with pre-gestational maternal stress exposure. As previously mentioned, although females seem particularly sensitive to the effects of perinatal fluoxetine exposure, male offspring appear particularly sensitive to maternal stress and depression. [START_REF] Gemmel | under review Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: interactions with maternal stress[END_REF] For example, pre-adolescent males, but not females, exposed to perinatal fluoxetine show reductions in pre-synaptic density and immature neurons in the dentate gyrus following pre-gestational maternal stress. [START_REF] Gemmel | under review Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: interactions with maternal stress[END_REF] Furthermore, SSRI exposure appears to affect the developmental trajectory of synaptic protein density, with adult female, but not male, offspring exposed during lactation/suckling to SSRIs showing significantly reduced pre-synaptic density in the dentate gyrus. [START_REF] Rayen | Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-dependent effects on hippocampal plasticity[END_REF] This enduring effect of SSRIs on synaptic proteins extends recent work showing that perinatal fluoxetine exposure can reduce perineuronal nets in the CA1 and DG region of juvenile mice hippocampi. [START_REF] Umemori | Distinct effects of perinatal exposure to fluoxetine or methylmercury on parvalbumin and perineuronal nets, the markers of critical periods in brain development[END_REF] In addition, postnatal fluoxetine treatment significantly alters gene expression related to hippocampal synaptic functioning and neurogenesis in selectively bred "Low Responder" rats predisposed for increased anxiety and behavioral abnormalities. [START_REF] Glover | Early-life exposure to the SSRI paroxetine exacerbates depression-like behavior in anxiety/depression-prone rats[END_REF] Thus, there is a likely role for perinatal fluoxetine exposure in altering hippocampal synaptic plasticity during development, however these effects of sexually differentiated and age-dependent (Table 1).

SSRIs and sex effects on hippocampal plasticity

As mentioned above, there is a persistent effect of sex in mediating perinatal SSRI exposure effects on hippocampal plasticity in adulthood. For example, adult female offspring, but not male offspring, exposed early in life to SSRIs have increased new cell survival, decreased BDNF mRNA expression, and reductions in hippocampal synaptophysin density in the granule cell layer. [START_REF] Boulle | Developmental fluoxetine exposure increases behavioral despair and alters epigenetic regulation of the hippocampal BDNF gene in adult female offspring[END_REF][START_REF] Boulle | Prenatal stress and early-life exposure to fluoxetine have enduring effects on anxiety and hippocampal BDNF gene expression in adult male offspring[END_REF][START_REF] Rayen | Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-dependent effects on hippocampal plasticity[END_REF] Others have shown that maternal postnatal fluoxetine treatment increases adult male immature neuron density in the hippocampus via doublecortin expression, while reducing such density in the adult female hippocampus. [START_REF] Gobinath | Maternal postpartum corticosterone and fluoxetine differentially affect adult male and female offspring on anxiety-like behavior, stress reactivity, and hippocampal neurogenesis[END_REF] Changes in steroid hormone levels during puberty likely contribute to sex-dependent SSRI sensitivity and structural changes in the hippocampus, as a result of altered estrogen and androgen receptor expression. [START_REF] Galea | Gonadal hormone modulation of hippocampal neurogenesis in the adult[END_REF][START_REF] Tabori | Ultrastructural evidence that androgen receptors are located at extranuclear sites in the rat hippocampal formation[END_REF][START_REF] Weiland | Distribution and hormone regulation of estrogen receptor immunoreactive cells in the hippocampus of male and female rats[END_REF] Serotonin plays a key role in sexual differentiation through its role in the development of the hypothalamic-pituitary-gonadal (HPG) axis. [START_REF] Dohler | Influence of neurotransmitters on sexual differentiation of brain structure and function[END_REF][START_REF] Jarzab | Serotoninergic influences on sexual differentiation of the rat brain[END_REF] The inhibition of the natural drop in serotonin in the first week of life resulting from SSRI exposure, likely antagonizes the perinatal masculinization effects of testosterone during the second and/or third week postpartum and alters feminization of the brain. Previous work shows that postnatal stimulation of serotonin synthesis, by injection of L-tryptophan, inhibits female sexual behavior and has an inhibitory effect on postnatal "organization" of female sexual behavior as well as on "activation" of female sexual behavior in adulthood. [START_REF] Dohler | Influence of neurotransmitters on sexual differentiation of brain structure and function[END_REF][START_REF] Jarzab | Serotoninergic influences on sexual differentiation of the rat brain[END_REF] In addition, a de-feminization of sexually dimorphic brain structures in females results from early-life stimulation of the serotonin synthesis [START_REF] Wilson | The effect of neonatal manipulation of hypothalamic serotonin levels on sexual activity in the adult rat[END_REF] and, conversely, treatment with parachlorophenylalanine, a serotonin synthesis inhibitor, enhances masculinization and defeminization. [START_REF] Wilson | The effect of neonatal manipulation of hypothalamic serotonin levels on sexual activity in the adult rat[END_REF][START_REF] Rayen | Developmental fluoxetine exposure and prenatal stress alter sexual differentiation of the brain and reproductive behavior in male rat offspring[END_REF] Therefore, sexually differentiated effects of perinatal SSRIs on hippocampal plasticity may be a result of changes to the serotonergic system early in life, as serotonin plays a role in sexual differentiation of the brain via development of the hypothalamic-pituitary-gonadal axis. [START_REF] Dohler | Influence of neurotransmitters on sexual differentiation of brain structure and function[END_REF][START_REF] Jarzab | Serotoninergic influences on sexual differentiation of the rat brain[END_REF]

The role of maternal stress

As shown above, the effects of perinatal SSRIs can differ in the presence of maternal stress in some regards; the effects of maternal stress are more pronounced and enduring than the effects of SSRIs. For example, maternal stress, regardless of perinatal SSRI exposure, significantly reduces pre-synaptic densities in pre-adolescent male, but not female, offspring and decreases the number of immature neurons in the granule cell layer of dentate gyrus in pre-adolescent and adult male, but not female, offspring. [START_REF] Rayen | Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-dependent effects on hippocampal plasticity[END_REF] Interestingly, pre-gestational maternal stress effects on offspring are in agreement with a number of studies showing that perinatal maternal stressors reduce hippocampal neurogenesis and plasticity later in life, particularly in males. [START_REF] Maccari | The consequences of early-life adversity: neurobiological, behavioural and epigenetic adaptations[END_REF][START_REF] Mirescu | Stress and adult neurogenesis[END_REF][START_REF] Lucassen | Prenatal stress reduces postnatal neurogenesis in rats selectively bred for high, but not low, anxiety: possible key role of placental 11beta-hydroxysteroid dehydrogenase type 2[END_REF] In line with this, an increasing amount of clinical research is reporting that child development outcomes are often more affected by maternal depression and associated risk factors prior to conception than by perinatal SSRI exposure. [START_REF] Oberlander | Disentangling Maternal Depression and Antidepressant Use During Pregnancy as Risks for Autism in Children[END_REF][START_REF] Brown | Association Between Serotonergic Antidepressant Use During Pregnancy and Autism Spectrum Disorder in Children[END_REF][START_REF] Mezzacappa | Risk for Autism Spectrum Disorders According to Period of Prenatal Antidepressant Exposure: A Systematic Review and Meta-analysis[END_REF] Thus, a mother's level of stress and depression during the perinatal period and prior to gestation can have a long-term impact on neurobehavioral outcomes in offspring, regardless, or in addition to, of perinatal SSRI effects. More research is needed to investigate additional therapeutic approaches which treat the effects of perinatal depression in both mothers and offspring.

Summary and Future directions

It is clear the perinatal SSRIs can have a long-term effect on plasticity in the hippocampus of exposed offspring. SSRIs can, at times, protect against the effect of maternal stress, have effects independent of maternal stress, and have effects on offspring that are sexually differentiated. The effects of perinatal SSRIs on hippocampal plasticity may be both direct, due to perinatal drug exposure, and indirect, with effects mediated by maternal care as well as SSRI effects on the placenta. [START_REF] Gemmel | Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: Interactions with pre-gestational maternal stress[END_REF][START_REF] Laurent | Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase[END_REF][START_REF] Muller | Impact of Maternal Serotonin Transporter Genotype on Placental Serotonin, Fetal Forebrain Serotonin, and Neurodevelopment[END_REF][START_REF] Bonnin | Fetal, maternal, and placental sources of serotonin and new implications for developmental programming of the brain[END_REF] Apart from effects on hippocampal plasticity, a growing body of literature shows that perinatal exposure to SSRIs affect a number of neuroendocrine systems and neurobehavioral outcomes which may be linked to the hippocampus. For example, developmental exposure to SSRIs alters the hypothalamic-pituitary-gonadal axis, [START_REF] Rayen | Developmental fluoxetine exposure and prenatal stress alter sexual differentiation of the brain and reproductive behavior in male rat offspring[END_REF][START_REF] Rayen | Developmental fluoxetine exposure facilitates sexual behavior in female offspring[END_REF] the hypothalamic-pituitary adrenal axis, [START_REF] Ishiwata | Selective serotonin reuptake inhibitor treatment of early postnatal mice reverses their prenatal stress-induced brain dysfunction[END_REF][START_REF] Pawluski | Developmental fluoxetine exposure differentially alters central and peripheral measures of the HPA system in adolescent male and female offspring[END_REF][START_REF] Pawluski | Prenatal SSRI exposure alters neonatal corticosteroid binding globulin, infant cortisol levels, and emerging HPA function[END_REF] and has been linked to poor social behaviors and increased mood disorders in children. [START_REF] Gemmel | Perinatal selective serotonin reuptake inhibitor medication (SSRI) effects on social behaviors, neurodevelopment and the epigenome[END_REF] However, there are also persistent effects of maternal stress and maternal mood on childhood outcomes, even with maternal treatment of SSRIs. [START_REF] Oberlander | Disentangling Maternal Depression and Antidepressant Use During Pregnancy as Risks for Autism in Children[END_REF][START_REF] Brown | Association Between Serotonergic Antidepressant Use During Pregnancy and Autism Spectrum Disorder in Children[END_REF][START_REF] Mezzacappa | Risk for Autism Spectrum Disorders According to Period of Prenatal Antidepressant Exposure: A Systematic Review and Meta-analysis[END_REF] What is abundantly clear is that untreated maternal depression is not an option for the mother or developing child. SSRIs are the first line treatment for maternal mood disorders and these medications are more effective if combined with psychotherapy. However, there are also a number of treatments available that may also be beneficial to the mother such as psychotherapy alone, parenting classes, exercise and diet change. [START_REF] Kim | Pharmacotherapy of postpartum depression: an update[END_REF][START_REF] Bodnar | Nutrition and depression: implications for improving mental health among childbearing-aged women[END_REF][START_REF] Swain | Parent-child intervention decreases stress and increases maternal brain activity and connectivity during own baby-cry: An exploratory study[END_REF] Regardless, the goal is to effectively treat the mother as the risk of untreated maternal depression out way the risk of the SSRI treatment exposure. However, many pregnant women prescribed SSRIs also remain depressed and anxious and this is detrimental to the mother, child and family. Therefore, SSRI treatment for maternal affective disorders during pregnancy should be treated on an individualized basis, which takes into account depression severity, likelihood of treatment response and probability of adverse fetal effects, and individual patient values and health. [START_REF] Angelotta | Treating Depression during Pregnancy: Are We Asking the Right Questions?[END_REF] The data reviewed here suggest that future treatment for maternal affective disorders, may also need to consider offspring sex, as well as SSRI timing and dosage.

Unfortunately, to date, there is very little research on the neurobiology of maternal mental illness even though 1 in 7 women suffering from perinatal depression or clinical levels of anxiety postpartum. [START_REF] Pawluski | The Neurobiology of Postpartum Anxiety and Depression[END_REF] Thus, more research is needed to understand the mechanisms behind maternal mental illness in order to develop effective and safe treatments for these disorders. Only then will we be able to improve the health and well-being of the mother and child.

Figure 1 .

 1 Figure 1. Photomicrographs of A) synaptophysin-immunoreactivity (ir) in the hippocampus (2x) and B) immature neurons in the granule call layer (doublecortin-ir) (40x) of pre-adolescent rat offspring. CA=cornu ammonis, DG=dentate gyrus. The arrows indicate immature neurons. Scale bar = 200µm (A) and 10µm (B).

Table 1 . Summary of rodent research investigating how perinatal SSRIs affect the developing hippocampus. P=postnatal day, G=gestation day, s.c.=subcutaneous, i.p.=intraperitoneal, pSSRI=perinatal SSRI, M=male, F=female.

 1

	Age studied Sex	Species	SSRI (dose/day)	pSSRI days admin	Results	SSRI admin method	Reference
	Hippocampus Serotonergic System
	P7, P21,	M	C57bl/6J	Fluoxetine	P1-21	-Fluox increases 5-HT at postnatal week 3 and 6	Oral administration to	Ishiwata et al
	P42, and		mice	(5mg/kg)		regardless of prenatal maternal stress.	offspring; Prenatal	2005
	P63					-Fluox restores 5-HIAA/5-HT in prenatally stressed	restraint stress
						offspring at postnatal week 3.
	P130	M	Long Evans	Citalopram	P8-21	-Citalopram decreases rate-limiting serotonin	s.c. injections to	Maciag et al
			rat	(10mg/kg)		enzyme, tryptophan hydroxylase, in dorsal raphe.	offspring	2006
	P21 and	M	Sprague-	Fluoxetine	P2-21	-Fluox restored reduction in ventral 5-HT following	Administration via	Nagano et al
	P84		Dawley rat	(Approximately		prenatal DEX at postnatal week 3.	drinking water to	2015
				17mg/kg)			dams; Prenatal
							dexamethasone
							(DEX)
	P22	M/F	Sprague-	Fluoxetine	P1-21	-Fluox reverses effects of prenatal maternal stress 5-	Osmotic minipump to	Gemmel et al
			Dawley rat	(5mg/kg)		HT and 5-HIAA levels and altered the dopaminergic	the dam; Prenatal	2016
						system.	restraint stress
	P27-30	M/F	Sprague-	Fluoxetine	G10-	-Fluox increases 5-HT and decreases 5-HIAA/5-HT	Oral biscuit	Gemmel et al
			Dawley rat	(10mg/kg)	P21	in all offspring and reverses effects of prenatal	administration to	2017
						maternal stress on 5-HT in female offspring.	dam; Pre-gestational
						-Pre-gestational stress, regardless of fluoxetine,	maternal stress
						decreased 5-HT, particularly in female offspring.
	Hippocampal Plasticity
	P21	?	Sprague-	Fluoxetine	P14-21	-Fluox increased BrdU cells and prevented apoptosis	i.p. injections to
			Dawley rat	(5mg/kg)		(via TUNEL-positive cells) in the dentate gyrus of	offspring; maternal
						maternally separated pups.	separation

JLP is funded by a Brain & Behavior Foundation NARSAD Young Investigator Grant.