The tributyltin leads to obesogenic mammary gland abnormalities in adult female rats

Accepted Manuscript Title: The Tributyltin Leads to Obesogenic Mammary Gland Abnormalities in Adult Female Rats Authors: Charles S. da Costa, Leandro Miranda-Alves, Michele A. La Merrill, Ian V. Silva, Jones B. Graceli PII: DOI: Reference:

S0378-4274(19)30053-0 https://doi.org/10.1016/j.toxlet.2019.02.016 TOXLET 10426

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

4 December 2018 31 January 2019 28 February 2019

Please cite this article as: da Costa CS, Miranda-Alves L, La Merrill MA, Silva IV, Graceli JB, The Tributyltin Leads to Obesogenic Mammary Gland Abnormalities in Adult Female Rats, Toxicology Letters (2019), https://doi.org/10.1016/j.toxlet.2019.02.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 The Tributyltin Leads to Obesogenic Mammary Gland Abnormalities in Adult Female Rats Charles S. da Costa1, Leandro Miranda-Alves2,3, Michele A. La Merrill4, Ian V. Silva1, Jones B. Graceli1 1

Dept of Morphology, Federal University of Espírito Santo, Brazil. 2Laboratory of Experimental

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Endocrinology, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Brazil.3Postgraduate Program in Endocrinology, Faculty of Medicine and Postgraduate Program

of Pharmacology and Medicinal Chemistry, Federal University of Rio de Janeiro, Brazil.4Dept of

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Environmental Toxicology, University of California at Davis, USA.

Author names and affiliations:

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Charles S. da Costa Dept of Morphology, Federal University of Espírito Santo, Brazil Email: [email protected]

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Leandro Miranda-Alves Experimental Endocrinology Research, Development and Innovation Group, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Brazil.Postgraduate Program in Endocrinology, School of Medicine, Federal University of Rio de Janeiro, Brazil. Email: [email protected]

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Michele A. La Merrill Dept of Environmental Toxicology, University of California at Davis, USA Email: [email protected]

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Ian V. Silva Dept of Morphology, Federal University of Espírito Santo, Brazil Email: [email protected] Jones B. Graceli Dept of Morphology, Federal University of Espírito Santo, Brazil Email: [email protected]

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*Corresponding author: Prof. Dr. Jones Bernardes Graceli Laboratório de Endocrinologia e Toxicologia Celular, Departamento de Morfologia/CCS, Universidade Federal do Espírito Santo. Av. Marechal Campos, 1468, Prédio do básico I, sala 5, 290440-090 Vitória, ES, Brasil. Tel.: +55-27-33357540/ 7369; Fax: +55-27-33357358. E-mail: [email protected]

2 Abbreviated title/ capsule: TBT induces rat mammary gland abnormalities

Conflict of interest statement The authors declare that there are no conflicts of interest related to this work.

Number of words: 5967 Number of figures: 5

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Number of supplemental figures: 3 Number of tables: 2

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Number of pages: 16

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Graphical abstract

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Highlights

-TBT disrupted the normal morphology of the mammary gland in adult female rats.

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-TBT leads to abnormal mammary gland fat accumulation by PPARγ expression. -Abnormal mammary gland fat accumulation by TBT may be associated with other mammary

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Abstract

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gland irregularities.

Tributyltin chloride (TBT) is an obesogen associated with several complications. However, few investigations have evaluated TBT effects on adult mammary glands (MG). In this investigation, we assessed whether TBT’s obesogenic effects resulted in abnormal MG fat pad expansion and

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other irregularities. TBT was administered to female rats (100ng/kg/day for 15 days via gavage), and their MG morphophysiological development was assessed. We further assessed the MG fat pad for PPARγ, ERα, and aromatase protein expression, as well as inflammation, oxidative stress (OS), apoptosis and fibrosis. Irregular MG morphological development such as lower TEB number, alveolar (AB), lobule and differentiation (DF) score were observed in TBT rats. TBT rats had abnormal MG fat accumulation as evidenced by increased numbers of hypertrophic adipocytes, triglyceride (TG) levels and PPARγ expression. A strong negative correlation

4 between the MG obesogenic makers and TEB number, AB and DF score were observed in TBT rats. MG inflammation was observed in TBT rats. A positive correlation between the MG obesogenic markers and inflammation were observed. High ERα and aromatase expression were observed in MG of TBT rats. MG OS, apoptosis and fibrosis were present in the TBT rats. Additionally, a positive correlation between the MG obesogenic markers and OS were observed in TBT rats. Thus, these data suggest that obesogenic TBT effects led to MG irregularities in the

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adult female rats.

Keywords: Tributyltin chloride, endocrine-disrupting chemicals, mammary gland fat pad,

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inflammation, ERα/aromatase, oxidative stress.

5 1. Introduction The mammary gland (MG) or breast is an apocrine gland only present in mammals which has a key offspring nourishment role through its milk synthesis and release (Inman et al., 2015; Sakakura et al., 1987). Both genders have MG with the same structure at birth, which begins branching into the fat pad as the female reaches puberty in response to sex hormonal cues. During the female lifetime, MG undergoes many physiologic changes, such as cyclic expansions corresponding to the reproductive cycle hormonal variations, as well as the changes that occur in

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pregnancy, lactation and involution (Lyons et al., 1958; Macias and Hinck, 2012). MG is comprised of an epithelial ductal tree embedded within a stromal fat pad with cell types including adipocytes, fibroblasts, and immune cells, that interact with each other to maintain its proper

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function (Howard and Gusterson, 2000). In puberty, MG ducts undergo branching, generating a

ductal tree that invades the fat pad. Ductal elongation is driven by cap cell proliferation located at the tips of the terminal end buds (TEBs) (Visvader and Stingl, 2014).

Recent investigations have reported that MG physiology is affected by environmental

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factors, as endocrine-disrupting chemicals (EDCs)(Kolla et al., 2017; Perrot-Applanat et al., 2018). EDCs can lead to abnormal MG morphological development, with/ without changing other

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puberty markers, sensitivity of carcinogens/xenobiotics/endogenous hormones and/or growth

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factors, gender specific features, milk production, offspring nutrition, etc, and that could be

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transient and persistent effects depending on dose, exposure parameters and development critical periods (Fenton, 2006; Gore et al., 2015). Tributyltin (TBT) is a persistent organotin pollutant widely used in various agroindustry applications that has been identified as EDC, as well as an

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obesogen chemical (Grün et al., 2006). TBT has several toxicologic effects on both invertebrate and vertebrate endocrine systems. TBT induces the imposex, the irregular induction of male sex

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characteristics in female gastropod mollusks leading to improper reproductive function (Fent, 1996; Grün et al., 2006).

Several studies have reported that consumption of seafood containing OTs, such as TBT

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is the primary source of human exposure in Asia, Europe and America (Toyoda et al., 2000; Fernandez et al., 2005; Rantakokko et al., 2006; Merlo et al., 2018). OTs are detected in human blood at levels that range from 64 to 155 ng/mL, which leads to TBT tissue accumulation and irregularities development (Whalen et al., 1999). Our previous studies have reported an increase

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in blood and/or organs tin levels after 100ng/Kg/day TBT exposure for 15 days leading to metabolic and reproductive abnormalities in adult female rats (Bertuloso et al., 2015; Sena et al., 2017). A congenital cryptorchidism association with placental OTs levels (0.21-0.26 ng/g) in Danish newborn boys was observed (Rantakokko et al., 2013). In addition, placental TBT levels (0.32 ng/g) are associated with increased weight gain during first three 3 months of life in newborn boys from Finland (Rantakokko et al., 2014). TBT promotes adipogenesis/lipogenesis and perturbs key regulators of metabolic pathways in different models by altering RXR and PPARγ

6 activation (Grün et al., 2006; Shoucri et al., 2017). In our previous recent studies, TBT exposure induced the metabolic syndrome and abnormal ovarian adipogenesis in adult female rats by improper fat metabolic function, oxidative stress, inflammation, gene control, etc (de Araújo et al., 2018; Freitas-Lima et al., 2018). However, TBT obesogenic actions in the MG are not well understood. Recent investigations have supported the key roles of obesity, inflammation and oxidative stress in abnormal MG function (Bronw, 2014; Syam et al., 2016; Chung et al., 2017). In addition, toxic hepatic, renal, neural, adrenal, reproductive and thyroid effects of TBT exposure

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are also reported (Grün et al., 2006; Coutinho et al., 2016; Merlo et al., 2016; Sena et al., 2017; Merlo et al., 2018).

Since the discovery that TBT is an EDC, to date, only one study reported its obesogenic

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effect on MG from newborns (Grün et al., 2006). Thus, the TBT effects on the adult MG remain

unclear. In the present study, we hypothesized that TBT will lead to MG abnormalities, resulting from obesogenic effects. We analysed the key indicators of female rat MG competence including the MG development/morphology, fat pad, inflammation, estrogen receptor alpha (ERα) and

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aromatase expression, oxidative stress, apoptosis and fibrosis. Identifying the altered MG functions due to TBT substantially contributes to our continuously evolving understanding of the

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MG targets of EDCs.

7 2. Materials and Methods 2.1 Chemicals Tributyltin chloride (TBT, 96%, Sigma, St. Louis, Mo., USA) was used based on previous investigation (Merlo et al., 2016). 2.2 Experimental animals Adult female Wistar rats (12-week-old) were maintained in a controlled temperature between 23 – 25°C with a 12:12-hr light/dark cycle. Rat chow and filtered tap water were provided

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ad libitum. All of the protocols were approved by the Ethics Committee of Animals of the Federal University of Espírito Santo (Nº 50/2017). The rats were weighed and divided into the following two groups (day 0): 1) CON (CON, n=10) rats were treated daily with a vehicle (0.4 % ethanol)

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and 2) TBT (TBT, n=10) rats were treated daily with TBT (100 ng/kg/day) for 15 days by gavage.

Rats exposed to TBT are referred to as the TBT rats throughout this investigation. Animals were weighed (day 15), anaesthetised using ketamine and xylazine (90 mg/kg and 4.5 mg/kg, ip) prior to euthanasia by decapitation, and wet reproductive tract organ and mammary gland weights (wt)

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were obtained (Suppl. Fig. 2A and B, and Fig. 1E, n=6-7). Inguinal mammary glands (MG, fourth

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pairs) were dissected as previously described (Xu et al., 2018). TBT doses and oral route of exposure were selected by measuring the serum tin levels and comparing the current findings with

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our previous studies that demonstrated toxicity on metabolic and reproductive tissues (Bertuloso

2.3 Estrous cycle assessment

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et al., 2015; de Araújo et al., 2018; Sena et al., 2017; Freitas-Lima et al., 2018).

TBT leads to abnormal estrous cyclicity (Podratz et al., 2012). Thus, we assessed the

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estrous cycle (Nelson et al., 1982). Briefly, vaginal smears were obtained daily at 10:00 a.m. The smears were stained with haematoxylin and eosin (H&E) and assessed under a microscope. The

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estrous cycle stage was classified as proestrus (P), estrus (E), or metestrus-diestrus (M-D) based on the observed ratios of cornified epithelial cells, nucleated epithelial and polymorphonuclear leukocytes. The frequencies of the estrous cycles and the days spent in the different phases were

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compared between the experimental groups (n=6-8) (Suppl. Fig. 1A and B). 2.4 Morphological analysis TBT leads to reproductive tract abnormalities (Sena et al., 2017). However, few studies

evaluated the MG histopathological features after TBT exposure (Wester et al., 1990). Thus, MG

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morphological analyses were evaluated (n=4 rats). For a more sensitive assessment of the MG densities and branching complexities, a whole mount preparation was evaluated in the contralateral MG. Thus, MGs were fixed for 12 hrs and a regular carmine alum staining protocol was performed (Stanko and Fenton, 2017). MG terminal end buds (TEBs), alveolar buds type 1 (AB1), type 2 (AB2) and lobules (L) were assessed under a 4X light microscope (ICC50 HD Leica Microsystems) (de Assis et al., 2010). TEBs were considered the bulb-shaped structures at the tips of the MG ducts and branches (Paine and Lewis, 2017). One or two small buds in duct

8 were considered AB1, more than two buds was considered AB2 and a buds group was the lobule (Manral et al., 2016; Monsefi et al., 2015). A differentiation (DF) assessment was applied to MG AB1, AB2 and L (de Assis et al., 2010). MG epithelial parameters were measured using FIJI software (Image J). The parameters analysed were the MG longitudinal growth and epithelial area (MEA). MG longitudinal growth was defined as the distance in mm starting from the base of attachment of the epithelial tree to the most distal point of the ductal growth (Elongation). MEA was defined as the perimeter of the MG epithelium reported in mm2(Stanko et al., 2015).

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Additionally, the fourth MG from the animal left side was removed and fixed in PBSformalin. MG sections were stained with H&E and examined for morphological parameters according to the literature (Ding et al., 2013).

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2.5 Adipocyte morphology and triglyceride assessment

TBT is an obesogen chemical, leading to obesity and other metabolic complications (Grün et al., 2006). After MG H&E staining, the randomized digital images were obtained to evaluated MG white adipocyte (WA) morphology (60 adipocytes/animal in 40X objective). MG WA

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diameter and the number of adipocytes were determined. Briefly, the adipose diameter was determined as the mean of distance of the major and minor adipocyte diameters, and

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quantification of adipocytes was performed and expressed as the number per unit area (µm2)

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(Ludgero-Correia Jr et al., 2012). Histomorphometric analyses were evaluated in a blinded

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manner and examined under a light microscope (Olympus BX43 with Olympus Q-color 5 camera). In addition, MG triglyceride (TG) levels were measured using colorimetric kits (Bioclin®, MG, Brazil) (Lima et al., 2012).

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2.6 Inflammation assessment

TBT is associated with inflammatory events (Freitas-Lima et al., 2018). Thus, the number

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of MG mast cells in their ducts and WAT were evaluated (n=4). MG sections were stained with Alcian Blue standard protocol (Sigma). Each of these sections was used to obtain 20 photomicrographs under a light microscope (40X objective). The number of positively stained

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cells was expressed per unit area (mm2) (Bertuloso et al., 2015). In addition, neutrophils and macrophages number were indirectly measured by the myeloperoxidase (MPO) and n-acetyl-βD-glucosaminidase (NAG) activity assays (Sena et al., 2017; de Araújo et al., 2018).

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2.7 Immunoblotting assessment MG total protein levels were obtained (Bertuloso et al., 2015). Briefly, MG samples (n=4)

were loaded onto an SDS/PAGE gel to perform the immunoblotting analysis (Bio-Rad). The primary antibodies included anti-ED1 (MCA341GA, 1:500, Bio-Rad, INC), estrogen receptor alpha (ERα, sc7207; 1:500, SCBT, INC), CYP19 (sc374176; 1:500, SCBT, INC), PPARγ (sc7273, 1:500, SCBT), COL1A1 (sc-293182, 1:500, SCBT, INC), COL3A1 (sc-271249, 1:500, SCBT, INC), GP91-PHOX (sc-130543, 1:500, SCBT, INC), caspase-3 (sc-7148, 1:500, SCBT, INC) and anti-GAPDH (sc25778, 1:1250, SCBT, INC). ERα, caspase-3 and GAPDH proteins

9 were detected using a secondary anti-rabbit IgG alkaline phosphatase conjugate (sc-2007, 1:1000, SCBT, INC), ED1, CYP19, PPARγ, COL3A1, COL1A1 and GP91-PHOX proteins were detected using a secondary anti-mouse IgG alkaline phosphatase conjugate (A3562, 1:1000, Sigma). The blots for ED1, ERα, COL1A1, COL3A1, GP91-PHOX, CYP19, PPARγ, and their respective GAPDH control were visualized using a colour development reaction containing BCIP/NBT solution (sc24981, SCBT, INC). The protein bands were analysed by densitometry using ImageJ software. The relative expression levels were normalized by dividing the values of the protein of

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interest by the corresponding internal control values. The total protein level was performed at the LABIOM Laboratory, UFES, Brazil. 2.8 Oxidative stress assessment

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TBT abnormalities are coincident with oxidative stress (OS) (de Araújo et al., 2018). Thus, OS assessment was performed. MG cryosections (8 µm, n=6-8) were incubated with the O2−-sensitive fluorescent dye dihydroethidium (DHE) to detect the superoxide anion (O2-) levels. Images were obtained using a Leica microscope DM 2500 coupled with a camera (DFC 310 FX

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Leica Microsystems, 20X objective). The signal intensity was analysed in 20 sections (Merlo et al., 2016). In addition, MG samples were prepared for a reduced glutathione (GSH) and

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thiobarbituric reactive species (TBARS) quantification assay (Syam et al., 2017). The

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microscopic analyses and photomicrographs were performed while blinded at the LHMI

2.9 Collagen deposition assessment

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Laboratory, UFES, Brazil.

TBT leads to reproductive tract injury (de Araújo et al., 2018). Thus, MG collagen

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deposition was performed (n=4). MG sections were stained with Picro-sirius Red and used to obtain 20 photomicrographs under 40X objective. The results were expressed as a percentage of

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the marked area (Sena et al., 2017). 3.0 Statistical analysis

All of the data are reported as the mean ± SEM. To identify possible outliers in the data,

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two-sided Grubbs’ test was used. When Grubbs’ test identified one outlier, we used an adapted ROUT method to detect any outliers from that column of data and removed them according to the Q setting at 1% (alpha = 0.01). D’Agostino and Pearson omnibus tests were used to assess normality of the data and no transformations were needed. Comparisons between the groups were

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performed using Student’s t tests for Gaussian data. Additionally, for the non-Gaussian data, a Mann-Whitney test was used. To evaluate the relationship between the assessed parameters, Spearman’s or Pearson’s correlation was used if a non-Gaussian or Gaussian distribution, respectively, was detected. All correlations were obtained from paired animal values. Finally, when statistical significance was identified, we tested whether linear or nonlinear regression was better fitting. A two-way ANOVA was also used to assess the body weight values acquired throughout the treatment, followed of Bonferroni post-test. A value of p ≤ 0.05 was regarded as

10 statistically significant. Statistical analyses and graphical construction were performed using

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GraphPad Prism version 6.00 (La Jolla, CA, USA).

11 3.0 Results 3.1 TBT rats have abnormal estrous cycles The estrous cyclicity was evaluated. Vaginal smears were collected daily for 15 days and examined under a microscope to evaluate the estrous cycle stage (Suppl. Fig. 1). TBT rats displayed irregular and longer estrous cycles and spent more days in the metestrus-diestrus (MD, approximately 50% of the time) phase compared with the CON rats (p ≤ 0.05 and p ≤ 0.05, Suppl. Fig.1A-B).

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3.2 TBT rats have altered reproductive tissue mass To evaluate the effect of TBT on the biometry, the body weights (bw) and reproductive tract organ wet weights were assessed (Suppl. Fig. 1 and 2, n=6-8). The initial bw was similar

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between the experimental groups (Suppl. Fig. 1C, p ≥ 0.05). However, once rats began to be

exposed to TBT, they had a consistently higher bw compared with CON rats (Suppl. Fig. 1C, p ≥ 0.05). TBT rats had significantly lower ovarian and uterine weights compared with the CON rats (Suppl. Fig. 2A and B, n=6, p ≤ 0.05). The 4th mammary gland (MG) was collected to evaluate

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their weight (Fig. 1A, B, C, D) and TBT rats had a 25% increase in MG weight compared with CON rats, suggesting higher fat content (p ≤ 0.05, Fig. 1E).

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3.3 TBT rats have MG histomorphometrical abnormalities

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MG histomorphometric assessment was performed on whole mounted glands stained with carmine alum staining (Fig. 2 A-F). The number of MG terminal end buds (TEBs, approximately

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34.5% of reduction, p ≤ 0.01, Fig.2G), alveolar buds type 1 (AB1, ~ 40.4% of reduction, p ≤ 0.001, Fig.2H), type 2 (AB2, ~ 27.6% of reduction, p ≤ 0.01, Fig.2I) and lobules (L, ~ 38.5% of

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reduction, p ≤ 0.001, Fig. 2J) were lower in TBT rats compared with CON rats. Also, we used the MG AB1, AB2 and L to evaluate a differentiation (DF) score assessment (DF1 and DF2). TBT

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rats had a 35.2% reduction in DF1 compared with CON rats (p ≤ 0.001, Fig. 2K). No significant differences were observed in DF2, MEA or elongation between CON and TBT rats (p > 0.05, Fig. 2L, O and P).

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3.4TBT rats have MG WAT hypertrophy, high triglyceride levels and PPARγ expression MG H&E-stained sections were evaluated (Fig. 3). MG CON sections indicated a regular

morphology, such as MG lobules, stroma, ducts (D) and adipocyte size/number (Fig. 3A and A1). However, in general, the TBT MG exhibited less organization, the presence of MG ducts dilated,

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periductal stroma and hypertrophic white adipose tissue (WAT) with presence of inflammatory cells (asterisks) surrounded by collagen fibers (Fig. 3B and B1). White adipocyte (WA) number and diameter of MG WAT were assessed. A 36% decrease in the number (n= 4, p ≤ 0.0001, Fig. 3C) and a 28% raise in the diameter of MG adipocytes were found in TBT rats (n=4, p ≤ 0.0001, Fig. 3D). This hypertrophy was reflected by the 36% increase in MG triglyceride (TG) levels (n=4-5, p ≤ 0.05, Fig. 3E) and 38% increase in MG WAT PPARγ protein expression (n = 4, p < 0.05, Fig. 3F) in TBT rats compared with CON rats.

12 3.5 TBT increased a MG ERα and CYP19 protein expression MG ERα and CYP19 protein expression were evaluated using immunoblotting analysis. A 244% increase in the ERα protein expression was observed in the MG from TBT rats compared with the CON rats (n=4, p ≤ 0.05, Fig. 3O). CYP19 protein expression was increased 40% in MG from TBT rats compared with CON rats (n = 4, p < 0.05, Fig. 3P). 3.6 TBT rats have MG inflammation MG inflammation was assessed by Alcian Blue staining (mast cells), MPO (neutrophils)

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and NAG (macrophage) activity and ED1 (macrophage) immunoblotting. An increase in the mast cell number were observed in the MG duct (~19.8% increase, n=4, p ≤ 0.05, Fig. 3G, H and K)

and WAT of TBT rats compared with the CON rats (~28.3% increase, n=4, p ≤ 0.05, Fig. 3I, J,

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and L). An increase in the MG NAG activity was identified in the TBT rats (~ 46.9% increase, p ≤ 0.001, Fig. 3M). Additionally, an increase in the MG ED1 protein expression was observed in the TBT rats (~ 36% increase, n=4, p ≤ 0.05, Fig. 3N). However, no significant change was observed in the MG MPO activity between the experimental groups (n=5, p > 0.05, Suppl. Fig.2

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C). 3.7 TBT leads to MG oxidative stress

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MG oxidative stress was assessed by DHE (production of O2− ), TBARS, GSH and GP91-

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PHOX immunoblotting assays (Fig.4). A 31% increase in the MG O2− levels was observed in TBT rats (n=5, p ≤ 0.01,Fig. 4A-C). Increased MG TBARS levels were observed in the TBT rats

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(~57.4% increase, n=5, p ≤ 0.05, Fig. 4D). However, GSH levels were 23% lower in the MG of TBT rats compared to CON rats (n=5, p ≤ 0.05, Fig. 4E). No significant difference was observed

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in MG GP91-PHOX protein expression between the experimental groups (n=4, p > 0.05, Fig. 4F).

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3.8 TBT leads to MG apoptosis

An assessment of the MG apoptosis was performed using caspase-3 immunoblotting (Fig. 4). An increase in the MG caspase-3 protein expression was observed in the TBT rats compared

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with the CON rats (CON: 1.00 ± 0.91; TBT: 1.38 ± 0.12, n=4, p ≤ 0.05, Fig. 4G). 3.9TBT leads to MG fibrosis An assessment of the MG collagen deposition was evaluated using Picro-sirius Red

staining and COL1A1 and COL3A1 immunoblotting (Fig. 5). MG collagen deposition was 90%

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increase in TBT rats (p ≤ 0.05, Fig. 5A, A1, B, B1, C). Additionally, an increase in the MG COL1A1 and COL3A1 protein expression was observed in the TBT rats compared with the CON rats (COL1A1: ~35% increase; COL3A1: ~60% increase, n=4, p ≤ 0.05, Fig. 5D-E). 4.0 Correlation among MG adipogenic markers, weight, development (TEBs, Differentiation score), inflammation, oxidative stress and fibrosis To evaluate the relationship between the obesogenic, e.g. MG weight, MG adipocyte diameter (WA), MG triglyceride content (TG), MG PPARγ content, reproductive toxicities, e.g.

13 TEBs, AB1, AB2, Lobule (L) and differentiation (DF) score, MG ERα content, MG aromatase content (CYP19) (Table 2) and other MG abnormalities, e.g. MG inflammation, MG oxidative stress (OS), and MG fibrosis of TBT (Suppl. Fig.3), pairwise correlation analyses were performed, and a linear fit was plotted. Several obesogenic and reproductive toxicities were correlated with each other (Table 2). We observed a positive linear correlation was observed between the MG WA diameter and weight (Pearson r: 0.72; p = 0.04, data now shown). We also observed a negative and positive linear

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correlation was observed between the MG WA diameter and AB2 score (p = 0.054) and ERα protein expression, respectively (p = 0.007). However, no significant linear correlation was

observed between the MG TEB numbers (p = 0.202), AB1 (p = 0.187), L (p = 0.169) and DF (p

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= 0.077) score, CYP19 protein expression (p = 0.228) and MG WA diameter (Table 2).

MG TG levels were highly correlated with several MG phenotypes important to reproductive fitness (Table 2). For example, MG TG levels were negatively correlated to the number of TEBs (p = 0.004), AB1 (p = 0.013), L (p = 0.027) and the DF score (p = 0.015). No ERα protein expression (p= 0.190) and MG TG levels.

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significant correlation was observed between the AB2 score (p= 0.170), CYP11A1 (p= 0.211),

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MG PPARγ protein expression was negatively correlated to AB1 score (p = 0.007). No

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significant correlation was observed between TEB number (p = 0.338), AB2 (p = 0.626), L (p = and MG PPARγ protein expression.

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0.503) and DF score (p = 0.163), CYP11A1 (p = 0.142) and ERα protein expression (p = 0.376)

Several reproductive toxicities were correlated with each other (Table 2). CYP19 protein

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expression was negatively correlated to AB1 (p = 0.045), AB2 (p = 0.054) and DF score (p = 0.031). No significant correlation was observed with TEB number (p = 0.256) and L score (p = 0.144) with CYP19 protein expression. No significant correlation with a linear association was

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observed between the ERα protein expression (p = 0.360), AB1 (p = 0.121), AB2 (p = 0.177), L (p = 0.438) and DF score (p = 0.153) and TEB numbers.

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MG WA diameter was positively correlated with inflammation (Pearson r: 0.71; p= 0.04

Suppl. Fig 3A), OS (Pearson r: 0.81; p= 0.01 Suppl. Fig 3B) and fibrosis (Pearson r: 0.87; p= 0.005 Suppl. Fig 3C). MG TG levels were highly correlated with inflammation (Pearson r: 0.83; p= 0.01 Suppl. Fig 3D) and OS (Pearson r: -0.79; p= 0.02 Suppl. Fig 3E), but no significant

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correlation with a linear association was observed with MG fibrosis (Suppl. Fig 3F). Inflammation was positively correlated with MG PPARγ protein expression (Pearson r: 0.80; p= 0.01, Suppl. Fig.3G), but not with OS and fibrosis (Suppl. Fig 3H and I). No significant correlation with a linear association was observed between CYP19 protein expression and MG inflammation, OS stress and fibrosis (Suppl. Fig 3J, K and L). A positive correlation with a linear association was observed between the ERα protein expression and MG OS (Pearson r: 0.84; p= 0.008, Suppl.

14 Fig.3N) and fibrosis (Pearson r: 0.91; p= 0.002, Suppl. Fig. 3O), but not with MG inflammation

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(Pearson r: -0.68; p= 0.06, Suppl. Fig. 3M).

15 4.0 Discussion Our study provides evidence for the first time that TBT exposure leads to mammary gland (MG) abnormalities as a result of, at least in part, its obesogenic effect in MG fat (white adipose tissue-WAT) of adult female rats. Irregular MG morphology/development with lower TEB number, lobule (L) and differentiation (DF) score were observed in TBT rats. Abnormal MG fat accumulation with fewer and wider adipocytes, containing more triglycerides (TG) level and PPARγ protein expression

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were observed in MG fat of TBT rats. A strong positive correlation between the MG adipocyte diameter and fat weight was observed in TBT rats. A strong negative correlation between the MG

TG levels, and numerous epithelial parameters, e.g. TEB number, AB1, L and DF score, were

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observed in TBT rats. These MG fat abnormalities were responsible for inflammation (presence

of mast cells and macrophage). A positive correlation between the MG adipocyte diameter, TG levels, PPARγ protein expression and inflammation was also observed. High ERα and CPY19 (aromatase) protein expression were also observed in MG of TBT rats. MG oxidative stress (OS)

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and apoptosis were present in the TBT rats. Additionally, a positive correlation between the MG adipocyte diameter, TG levels and OS were observed. After these MG complications, MG

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remodelling was observed by an increase in MG fibrosis in TBT rats. Thus, these data suggest

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that obesogenic TBT effects in MG fat led to MG irregularities in the adult female rats. From previous investigations, we learned that TBT is an obesogenic chemical (EDC

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subclass), able to increase obesity and metabolic complications by abnormal RXR, PPARγ activation and abnormal modulation of other signaling networks (de Araújo et al., 2018; Freitas-

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Lima et al., 2018; Grün et al., 2006; Shoucri et al., 2017). An increase in fat deposition is a common feature after TBT exposure in different mammalian models (Bertuloso et al., 2015; Grün

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et al., 2006; Penza et al., 2011). For example, at a TBT exposure as low as100 ng/kg/day(TBT for 15 days), female rats had increased fat mass (Coutinho et al., 2016; de Araújo et al., 2018; Merlo et al., 2016). Our results are consistent with these previous data, which demonstrated increase in

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MG/inguinal fat pad weight, suggesting TBT consistently increases fat mass accumulation without significant change in body weight. Several studies of different EDCs reported they affect MG epithelial and fat cell

development in rodent (La Merrill et al., 2009; Vandenberg et al., 2012). However, there are few

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studies that evaluated OTs effects on MG using in vitro and in vivo models (Balas et al., 2011; Cardarelli et al., 1984; Grün et al., 2006; Silva et al., 2013) (Table 1). In addition, we did not find a similar study using TBT exposure and evaluating MG morphology and development parameters in vivo. For this reason, we compared the MG effect found in our model with other studies that used another obesogenic chemical, such as BPA, that also activated the PPARγ signaling similar to TBT action (Grün et al., 2006; Heindel et al., 2015). Low-dose gestational or perinatal obesogen bisphenol A (BPA, 25 ng/kg/day) exposure to mice caused a stimulation of MG growth,

16 with increased TEB early and more lateral branches/ gland in the adult. However, a BPA higher dose (250 ng/kg/day) had an opposite effect, reducing lateral and longitudinal MG growth (Markey et al., 2001; Muñoz-de-Toro et al., 2005). These high BPA dose effects are similar to our study of low TBT dose effects, where we observed a reduction in TEB number, AB1, AB2, Lobule and DF 1 score in TBT rats, showing atrophy in MG structures. Given TBT also increased ERα and aromatase protein expression at the conclusion of our study, one may have anticipated a MG epithelium growth promotion instead (Mueller et al., 2002; Mulac-Jericevic et al., 2003).

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However, the abnormal reproductive cycling with excess days in metestrus-diestrus throughout the study in TBT rats is expected to lower circulating estrogen levels and could explain the

decreased MG epithelial growth (Macias and Hinck, 2012). Indeed, we recently showed that TBT

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exposure led to reduction in serum estrogen and progesterone levels, as well as an increase in testosterone levels in female rats (de Araújo et al., 2018; Sena et al., 2017).

From previous our investigations, we reported abnormal fat deposition with adipocyte hypertrophy, lipid profile and PPARγ expression after TBT exposure 100 ng/kg TBT for 15 days

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in female rats (Bertuloso et al., 2015; Coutinho et al., 2016; Freitas-Lima et al., 2018). In addition, we and other studies observed an irregular regulatory signalling with ectopic fat accumulation, as

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result of obesogenic TBT effect in different rodent tissues, such as liver, adrenal and ovary

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(Chamorro-García et al., 2013; Merlo et al., 2016; de Araújo et al., 2018). Our results are consistent with these previous data, which demonstrated MG adipocyte reduction number,

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adipocyte hypertrophy, high TG levels and PPARγ protein expression and a strong positive correlation between the MG adipocyte diameter and fat weight. Thus, our data suggested that

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TBT had obesogenic effect in MG fat tissue. Normal MG epithelia function, e.g. growth, differentiation, lactation or involution, has a

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close relationship with the surrounding depot of adipocytes in mammalian models (Hovey and Aimo, 2010; Sakakura et al., 1987). Both diet and exogenous chemicals have been shown to impact both the epithelium and the adipocytes of MG. For example, excessive MG adipose and

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TG levels were associated with impaired MG alveolar development and differentiation, lactogenesis and milk composition, as well as a reduced parenchymal mass in female mice with diet-induced obesity (Flint, 2005). Further, MG of rat dams treated with monosodium glutamate presented a high adipocyte content and reduced alveoli development (Cancian et al., 2016).

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Consistent with these previous data, we observed a strong negative correlation between the MG TG levels, TEB number, AB1, L and DF score in TBT rats that demonstrated the obesogenic TBT role in MG fat is responsible to their abnormalities in our model. TBT is able to induces inflammation in several tissues (Coutinho et al., 2016; Mitra et al., 2013). Abnormal fat expansion condition as result of TBT exposure is typically accompanied by low-grade inflammation, and an abnormal adipokine profile (Freitas-Lima et al., 2018). As we have indicated here and previously, TBT increased mast cell and macrophage number in

17 numerous tissues including adipose tissue (Bertuloso et al. 2015; Freitas-Lima et al. 2018). MG fat tissue is also susceptible to increase inflammation in obesity, as shown by relationship between presence of macrophage and MG fat adiposity (Subbaramaiah et al., 2011). Also, a higher NAG activity and ED1 protein expression (macrophage) were observed in MG from TBT rats. In addition, a positive correlation between the MG adipocyte diameter, TG levels, PPARγ protein expression and inflammation were observed in TBT rats. Other studies suggested that cytokines from inflamed MG obese fat tissue can stimulate estrogen production by aromatase activity,

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leading to MG abnormalities and increased risk of MG cancer (Brown, 2014; Harada et al., 1993). We observed a high MG ERα and CPY19 (aromatase) protein expression in TBT rats. A negative

correlation between CYP19 protein expression and MG AB and DF score was observed

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suggesting their role in abnormal MG morphology. A positive correlation between the MG adipocyte diameter and ERα protein expression were observed. Additionally, we observed a positive correlation between MG ERα protein expression, weight and OS (Suppl. Fig. 2). Thus, these data suggested that MG obesogenic TBT effect is associated with inflammation and

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abnormal ERα/CPY19 protein expression.

TBT exposure is associated with cellular dysfunction by OS in at least four tissues, e.g.

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vascular, renal, adrenal and pituitary (Coutinho et al., 2016; Merlo et al., 2016; Rodrigues et al.,

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2014). Our current study extends these previous findings to MG by demonstrating an increase in

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MG O2− and TBARS levels, as well as a reduction in MG GSH levels in TBT rats, suggesting a higher OS in MG from TBT rats. Additionally, a positive correlation between the MG adipocyte diameter, TG levels and OS were observed. As result of these abnormalities, we also observed a

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MG apoptosis by increase in MG caspase-3 protein expression in TBT rats. In addition, our previous studies showed that TBT induces cellular damage that can be

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replaced by fibrous tissue (de Araújo et al., 2018; Merlo et al., 2016). Thus, MG collagen deposition was assessed. In agreement with these previous findings, MG of TBT rats displayed an increase in collagen deposition, as well as a high COLA1 and COL3A1 protein expression.

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Thus, TBT is able to induce fibrosis in the MG. From previous studies, we learned that TBT is an obesogen chemical that increases

susceptibility to metabolic diseases, impairing adipocyte morphophysiology, leading to obesity and abnormal inflammation (Grün et al., 2006; Bertuloso et al., 2015; Sena et al., 2017).

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Inflammation in several experimental models, such as TBT exposure plays a critical role in obesity complications, such as abnormal adipogenesis, adipokine release, oxidative stress, apoptosis, fibrosis, etc which play an important role in metabolic control (Freitas-Lima et al., 2018; Pereira et al., 2012). In conclusion, our model demonstrates that TBT exposure leads to MG epithelial abnormalities as a result of, at least in part, its obesogenic effect in MG fat of adult female rats. TBT induced irregular morphology/development, which may be associated with high and

18 abnormal MG fat mass accumulation, such as high MG TG levels. Hypertrophic MG fat tissue with high TG levels and PPARγ expression were associated with inflammation, oxidative stress, apoptosis and fibrosis. Furthermore, the abnormal inflammation of MG fat could be associated with high ERα/CPY19 protein expression and other MG risks. This study increases our understanding of the obesogenic TBT effects on the MG may due to abnormal fat adipogenic markers regulation in adult female rats.

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5.0 Acknowledgements This research was supported by CNPq/FAPES 24/2018-PRONEX (#572/2018), FAPES Nº 03/2017-UNIVERSAL (#179/2017), FAPES (#72630477/2014), CNPq (#304724/2017-3/ Nº

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12/2017) and FAPERJ (E-26/201.520/2014; E-26/010.000175/2016). JBG awarded grants by FAPES, CNPq and CNPq/FAPES. LM-A awarded grants by FAPERJ. This study was financed

in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -

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Finance Code 00.

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6.0 Disclosure statement: the authors have nothing to disclose.

19 7.0 References: Balas, V.I., Verginadis, I.I., Geromichalos, G.D., Kourkoumelis, N., Male, L., Hursthouse, M.B., Repana, K.H., Yiannaki, E., Charalabopoulos, K., Bakas, T., Hadjikakou, S.K., 2011. Synthesis, structural characterization and biological studies of the triphenyltin(IV) complex with 2-thiobarbituric acid. Eur. J. Med. Chem. 46, 2835–2844. https://doi.org/10.1016/j.ejmech.2011.04.005

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Bertuloso, B.D., Podratz, P.L., Merlo, E., de Ara??jo, J.F.P., Lima, L.C.F., de Miguel, E.C., de Souza, L.N., Gava, A.L., de Oliveira, M., Miranda-Alves, L., Carneiro, M.T.W.D.,

Nogueira, C.R., Graceli, J.B., 2015. Tributyltin chloride leads to adiposity and impairs

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metabolic functions in the rat liver and pancreas. Toxicol. Lett. 235, 45–59. https://doi.org/10.1016/j.toxlet.2015.03.009

Brown, K.A., 2014. Impact of obesity on mammary gland inflammation and local estrogen production. J. Mammary Gland Biol. Neoplasia 19, 183–189.

U

https://doi.org/10.1007/s10911-014-9321-0

N

Cancian, C.R.C., Leite, N.C., Montes, E.G., Fisher, S.V., Waselcoski, L., Stal, E.C.L.,

A

Christoforo, R.Z., Grassiolli, S., 2016. Histological and metabolic state of dams suckling small litter or MSG-treated pups. Sci. World J. 2016, 1–12.

M

https://doi.org/10.1155/2016/1678541

Cardarelli, N.F., Cardarelli, B.M., Libby, E.P., Dobbins, E., 1984. © ORGANOTIN

ED

IMPLICATIONS IN ANTICARCINOGENESIS . EFFECTS OF SEVERAL ORGANOTINS ON TUMOUR GROWTH RATE IN MICE regimen is shown in Table 2 .

PT

Measurements were made externally and refer. Aust. J. Exp. Biol. Med. Sci. 62, 209–214. https://doi.org/10.1038/icb.1984.21

CC E

Chamorro-García, R., Sahu, M., Abbey, R.J., Laude, J., Pham, N., Blumberg, B., 2013. Transgenerational inheritance of increased fat depot size, stem cell reprogramming, and hepatic steatosis elicited by prenatal exposure to the obesogen tributyltin in mice. Environ. Health Perspect. 121, 359–366. https://doi.org/10.1289/ehp.1205701

A

Chung, H.H., Or, Y.Z., Shrestha, S., Loh, J.T., Lim, C.L., Ong, Z., Woo, A.R.E., Su, I.H., Lin, V.C.L., 2017. Estrogen reprograms the activity of neutrophils to foster protumoral microenvironment during mammary involution. Sci. Rep. 7, 46485. https://doi.org/10.1038/srep46485 Coutinho, J.V.S., Freitas-Lima, L.C., Freitas, F.F.C.T., Freitas, F.P.S., Podratz, P.L., Magnago, R.P.L., Porto, M.L., Meyrelles, S.S., Vasquez, E.C., Brandão, P.A.A., Carneiro,

20 M.T.W.D., Paiva-Melo, F.D., Miranda-Alves, L., Silva, I. V., Gava, A.L., Graceli, J.B., 2016. Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats. Toxicol. Lett. 260, 52–69. https://doi.org/10.1016/j.toxlet.2016.08.007 de Araújo, J.F.P., Podratz, P.L., Sena, G.C., Merlo, E., Freitas-Lima, L.C., Ayub, J.G.M., Pereira, A.F.Z., Santos-Silva, A.P., Miranda-Alves, L., Silva, I. V, Graceli, J.B., 2018. The obesogen tributyltin induces abnormal ovarian adipogenesis in adult female rats. Toxicol.

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Lett. 295, 99–114. https://doi.org/10.1016/j.toxlet.2018.06.1068 de Assis, S., Warri, A., Cruz, M.I., Hilakivi-Clarke, L., 2010. Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats. J. Vis. Exp. 21, 2–4.

SC R

https://doi.org/10.3791/2260

Ding, L., Zhao, Y., Warren, C.L., Sullivan, R., Eliceiri, K.W., Shull, J.D., 2013. Association of cellular and molecular responses in the rat mammary gland to 17β-estradiol with

susceptibility to mammary cancer. BMC Cancer 13, 573. https://doi.org/10.1186/1471-

U

2407-13-573

N

Fent, K., 1996. Ecotoxicology of organotin compounds. Crit. Rev. Toxicol. 26, 1–117.

A

Fenton, S.E., 2006. Endocrine-disrupting compounds and mammary gland development: Early

M

exposure and later life consequences. Endocrinology 147, 18–24. https://doi.org/10.1210/en.2005-1131

ED

Fernandez, M.A., Wagener, A.D.L.R., Limaverde, A.M., Scofield, A.L., Pinheiro, F.M., Rodrigues, E., 2005. Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil. Mar. Environ.

PT

Res. 59, 435–452. https://doi.org/10.1016/j.marenvres.2004.07.001 Flint, D.J., 2005. Diet-induced obesity impairs mammary development and lactogenesis in

CC E

murine mammary gland. AJP Endocrinol. Metab. 288, E1179–E1187. https://doi.org/10.1152/ajpendo.00433.2004

Freitas-Lima, L.C., Merlo, E., Campos Zicker, M., Navia-Pelaez, J.M., de Oliveira, M., dos

A

Santos Aggum Capettini, L., Nogueira, C.R., Versiani Matos Ferreira, A., Sousa Santos, S.H., Bernardes Graceli, J., 2018. Tributyltin impacts in metabolic syndrome development through disruption of angiotensin II receptor signaling pathways in white adipose tissue from adult female rats. Toxicol. Lett. 299, 21–31. https://doi.org/10.1016/j.toxlet.2018.08.018

Gore, A.C., Chappell, V.A., Fenton, S.E., Flaws, J.A., Nadal, A., Prins, G.S., Toppari, J., Zoeller, R.T., 2015. Executive Summary to EDC-2: The Endocrine Society’s second

21 Scientific Statement on endocrine-disrupting chemicals. Endocr. Rev. https://doi.org/10.1210/er.2015-1093 Grün, F., Watanabe, H., Zamanian, Z., Maeda, L., Arima, K., Cubacha, R., Gardiner, D.M., Kanno, J., Iguchi, T., Blumberg, B., 2006. Endocrine-Disrupting Organotin Compounds Are Potent Inducers of Adipogenesis in Vertebrates. Mol. Endocrinol. 20, 2141–2155. https://doi.org/10.1210/me.2005-0367

IP T

Harada, N., Utsumi, T., Takagi, Y., 1993. Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and

switching of tissue-specific exons 1 in carcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 90,

SC R

11312–6.

Heindel, J.J., Newbold, R., Schug, T.T., 2015. Endocrine disruptors and obesity. Nat. Rev. Endocrinol. 11, 653–661. https://doi.org/10.1038/nrendo.2015.163

U

Hovey, R.C., Aimo, L., 2010. Diverse and active roles for adipocytes during mammary gland

https://doi.org/10.1007/s10911-010-9187-8

N

growth and function. J. Mammary Gland Biol. Neoplasia 15, 279–290.

A

Howard, B.A., Gusterson, B.A., 2000. Human breast development. J. Mammary Gland Biol.

M

Neoplasia 5, 119–37.

Inman, J.L., Robertson, C., Mott, J.D., Bissell, M.J., 2015. Mammary gland development: cell

ED

fate specification, stem cells and the microenvironment. Development 142, 1028–1042. https://doi.org/10.1242/dev.087643

Kolla, S., Pokharel, A., Vandenberg, L.N., 2017. The mouse mammary gland as a sentinel

PT

organ: distinguishing ‘control’ populations with diverse environmental histories. Environ.

CC E

Heal. 16, 25. https://doi.org/10.1186/s12940-017-0229-1 La Merrill, M., Kuruvilla, B.S., Pomp, D., Birnbaum, L.S., Threadgill, D.W., 2009. Dietary fat alters body composition, mammary development, and cytochrome P450 induction after maternal TCDD exposure in DBA/2J mice with low-responsive Aryl hydrocarbon

A

receptors. Environ. Health Perspect. 117, 1414–1419. https://doi.org/10.1289/ehp.0800530

Lima, L.C.F., Porto, M.L., Campagnaro, B.P., Tonini, C.L., Nogueira, B. V, Pereira, T.M., Vasquez, E.C., Meyrelles, S.S., 2012. Mononuclear cell therapy reverts cuff-induced thrombosis in apolipoprotein E-deficient mice. Lipids Health Dis. 11, 96. https://doi.org/10.1186/1476-511X-11-96 Ludgero-Correia Jr, A., Aguila, M.B., Mandarim-de-Lacerda, C.A., Faria, T.S., 2012. Effects of high-fat diet on plasma lipids, adiposity, and inflammatory markers in ovariectomized

22 C57BL6 mice. Nutrition 28, 316–323. https://doi.org/:10.1016/j.nut.2011.07.014 Lyons, W.R., Li, C.H., Johnson, R.E., 1958. The hormonal control of mammary growth and lactation. Recent Prog. Horm. Res. 14, 219-48; discussion 248-54. Macias, H., Hinck, L., 2012. Mammary gland development. Wiley Interdiscip. Rev. Dev. Biol. 1, 533–557. https://doi.org/10.1002/wdev.35 Manral, C., Roy, S., Singh, M., Gautam, S., Yadav, R.K., Rawat, J.K., Devi, U., Ansari, M.N.,

IP T

Saeedan, A.S., Kaithwas, G., 2016. Effect of β-sitosterol against methyl nitrosourea-

induced mammary gland carcinoma in albino rats. BMC Complement. Altern. Med. 16, 260. https://doi.org/10.1186/s12906-016-1243-5

SC R

Markey, C.M., Luque, E.H., Munoz De Toro, M., Sonnenschein, C., Soto, a M., 2001. In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland. Biol. Reprod. 65, 1215–1223.

U

https://doi.org/10.1095/biolreprod.104.036582

N

Merlo, E., Podratz, P.L., Sena, G.C., de Araújo, J.F.P., Lima, L.C.F., Alves, I.S.S., Gama-deSouza, L.N., Pelição, R., Rodrigues, L.C.M., Brandão, P.A.A., Carneiro, M.T.W.D., Pires,

A

R.G.W., Martins-Silva, C., Alarcon, T.A., Miranda-Alves, L., Silva, I. V., Graceli, J.B.,

M

2016. The Environmental Pollutant Tributyltin Chloride Disrupts the HypothalamicPituitary-Adrenal Axis at Different Levels in Female Rats. Endocrinology 157, 2978–

ED

2995. https://doi.org/10.1210/en.2015-1896

Merlo, E., Silva, I. V., Cardoso, R.C., Graceli, J.B., 2018. The obesogen tributyltin induces features of polycystic ovary syndrome (PCOS): a review. J. Toxicol. Environ. Heal. Part B

PT

21, 181–206. https://doi.org/10.1080/10937404.2018.1496214 Mitra, S., Gera, R., Siddiqui, W.A., Khandelwal, S., 2013. Tributyltin induces oxidative

CC E

damage, inflammation and apoptosis via disturbance in blood–brain barrier and metal homeostasis in cerebral cortex of rat brain: An in vivo and in vitro study. Toxicology 310, 39–52. https://doi.org/10.1016/j.tox.2013.05.011

A

Monsefi, M., Abedian, M., Azarbahram, Z., Ashraf, M.J., 2015. Salvia officinalis L. induces alveolar bud growing in adult female rat mammary glands. Avicenna J. phytomedicine 5, 560–7.

Mueller, S.O., Clark, J.A., Myers, P.H., Korach, K.S., 2002. Mammary Gland Development in Adult Mice Requires Epithelial and Stromal Estrogen Receptor α. Endocrinology 143, 2357–2365. https://doi.org/10.1210/endo.143.6.8836 Mulac-Jericevic, B., Lydon, J.P., DeMayo, F.J., Conneely, O.M., 2003. Defective mammary

23 gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc. Natl. Acad. Sci. U. S. A. 100, 9744–9. https://doi.org/10.1073/pnas.1732707100 Muñoz-de-Toro, M., Markey, C.M., Wadia, P.R., Luque, E.H., Rubin, B.S., Sonnenschein, C., Soto, A.M., 2005. Perinatal Exposure to Bisphenol-A Alters Peripubertal Mammary Gland Development in Mice. Endocrinology 146, 4138–4147. https://doi.org/10.1210/en.20050340

IP T

Nelson, J.F., Felicio, L.S., Randall, P.K., Sims, C., Finch, C.E., 1982. A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology. Biol. Reprod. 27, 327–39. https://doi.org/10.1095/biolreprod27.2.327

SC R

Paine, I.S., Lewis, M.T., 2017. The Terminal End Bud: the Little Engine that Could. J.

Mammary Gland Biol. Neoplasia 22, 93–108. https://doi.org/10.1007/s10911-017-9372-0 Penza, M., Jeremic, M., Marrazzo, E., Maggi, A., Ciana, P., Rando, G., Grigolato, P.G., Di

U

Lorenzo, D., 2011. The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose.

N

Toxicol. Appl. Pharmacol. 255, 65–75. https://doi.org/10.1016/j.taap.2011.05.017

A

Pereira, S.S., Teixeira, L.G., Aguilar, E.C., Matoso, R.O., Soares, F.L.P., Ferreira, A.V.M.,

M

Alvarez-Leite, J.I., 2012. Differences in adipose tissue inflammation and oxidative status in C57BL/6 and ApoE-/- mice fed high fat diet. Anim. Sci. J. 83, 549–555.

ED

https://doi.org/10.1111/j.1740-0929.2011.00982.x Perrot-Applanat, M., Kolf-Clauw, M., Michel, C., Beausoleil, C., 2018. Alteration of mammary gland development by bisphenol a and evidence of a mode of action mediated through

PT

endocrine disruption. Mol. Cell. Endocrinol. https://doi.org/10.1016/j.mce.2018.06.015 Podratz, P.L., Filho, V.S.D., Lopes, P.F.I., Sena, G.C., Matsumoto, S.T., Samoto, V.Y., Takiya,

CC E

C.M., Miguel, E.D.C., Silva, I.V., Graceli, J.B., 2012. Tributyltin impairs the reproductive cycle in female rats. J. Toxicol. Environ. Heal. - Part A Curr. Issues 75, 1035–1046. https://doi.org/10.1080/15287394.2012.697826

A

Rantakokko, P., Kuningas, T., Saastamoinen, K., Vartiainen, T., 2006. Dietary intake of organotin compounds in Finland: A market-basket study. Food Addit. Contam. 23, 749– 756. https://doi.org/10.1080/02652030600779908

Rantakokko, P., Main, K.M., Wohlfart-Veje, C., Kiviranta, H., Airaksinen, R., Vartiainen, T., Skakkebæk, N.E., Toppari, J., Virtanen, H.E., 2014. Association of placenta organotin concentrations with growth and ponderal index in 110 newborn boys from Finland during the first 18 months of life: a cohort study. Environ. Heal. 13, 45.

24 https://doi.org/10.1186/1476-069X-13-45 Rantakokko, P., Main, K.M., Wohlfart-Veje, C., Kiviranta, H., Airaksinen, R., Vartiainen, T., Skakkebæk, N.E., Toppari, J., Virtanen, H.E., 2013. Association of placenta organotin concentrations with congenital cryptorchidism and reproductive hormone levels in 280 newborn boys from Denmark and Finland. Hum. Reprod. 28, 1647–1660. https://doi.org/10.1093/humrep/det040

IP T

Rodrigues, S.M.L., Ximenes, C.F., de Batista, P.R., Simões, F. V., Coser, P.H.P., Sena, G.C., Podratz, P.L., de Souza, L.N.G., Vassallo, D. V., Graceli, J.B., Stefanon, I., 2014.

Tributyltin contributes in reducing the vascular reactivity to phenylephrine in isolated

SC R

aortic rings from female rats. Toxicol. Lett. 225, 378–385. https://doi.org/10.1016/j.toxlet.2014.01.002

Sakakura, T., Kusano, I., Kusakabe, M., Inaguma, Y., Nishizuka, Y., 1987. Biology of

mammary fat pad in fetal mouse: capacity to support development of various fetal

U

epithelia in vivo. Development 100, 421–430.

N

Sena, G.C., Freitas-Lima, L.C., Merlo, E., Podratz, P.L., de Araújo, J.F.P., Brandão, P.A.A.,

A

Carneiro, M.T.W.D., Zicker, M.C., Ferreira, A.V.M., Takiya, C.M., de Lemos Barbosa, C.M., Morales, M.M., Santos-Silva, A.P., Miranda-Alves, L., Silva, I. V., Graceli, J.B.,

M

2017. Environmental obesogen tributyltin chloride leads to abnormal hypothalamicpituitary-gonadal axis function by disruption in kisspeptin/leptin signaling in female rats.

ED

Toxicol. Appl. Pharmacol. 319, 22–38. https://doi.org/10.1016/j.taap.2017.01.021 Shoucri, B.M., Martinez, E.S., Abreo, T.J., Hung, V.T., Moosova, Z., Shioda, T., Blumberg, B.,

PT

2017. Retinoid x receptor activation alters the chromatin landscape to commit mesenchymal stem cells to the adipose lineage. Endocrinology 158, 3109–3125.

CC E

https://doi.org/10.1210/en.2017-00348 Silva, A., Luís, D., Santos, S., Silva, J., Mendo, A.S., Coito, L., Silva, T.F.S., Guedes Da Silva, M.F.C., Martins, L.M.D.R.S., Pombeiro, A.J.L., Borralho, P.M., Rodrigues, C.M.P., Cabral, M.G., Videira, P.A., Monteiro, C., Fernandes, A.R., 2013. Biological

A

characterization of the antiproliferative potential of Co(II) and Sn(IV) coordination compounds in human cancer cell lines: A comparative proteomic approach. Drug Metabol. Drug Interact. 28, 167–176. https://doi.org/10.1515/dmdi-2013-0015

Stanko, J.P., Easterling, M.R., Fenton, S.E., 2015. Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts. Reprod. Toxicol. 54, 129–135. https://doi.org/10.1016/j.reprotox.2014.11.004

25 Stanko, J.P., Fenton, S.E., 2017. Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method. J. Vis. Exp. 2017, 1–12. https://doi.org/10.3791/55789 Subbaramaiah, K., Howe, L.R., Bhardwaj, P., Du, B., Gravaghi, C., Yantiss, R.K., Zhou, X.K., Blaho, V.A., Hla, T., Yang, P., Kopelovich, L., Hudis, C.A., Dannenberg, A.J., 2011. Obesity is associated with inflammation and elevated aromatase expression in the mouse mammary gland. Cancer Prev. Res. 4, 329–346. https://doi.org/10.1158/1940-6207.CAPR-

IP T

10-0381

Syam, S., Bustamam, A., Hashim, N.M., Ghaderian, M., Hobani, Y.H., Makeen, A.,

Abdelwahab, S.I., Mohan, S., 2017. Corrigendum to “β-Mangostin suppresses LA-7 cells

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proliferation in vitro and in vivo : Involvement of antioxidant enzyme modulation;

suppression of matrix metalloproteinase and α6β4 integrin signaling pathways” [J. Funct. Foods 22 (2016) 504–517]. J. Funct. Foods 34, 478–479.

U

https://doi.org/10.1016/j.jff.2017.05.030

N

Toyoda, M., Sakai, H., Kobayashi, Y., Komatsu, M., Hoshino, Y., Horie, M., Saeki, M., Hasegawa, Y., Tsuji, M., Kojima, M., Toyomura, K., Kumano, M., Tanimura, A., 2000.

A

Daily Dietary Intake of Tributyltin, Dibutyltin, Triphenyltin and Diphenyltin Compounds

M

According to a Total Diet Study in a Japanese Population. J. Food Hyg. Soc. Japan (Shokuhin Eiseigaku Zasshi) 41, 280–286. https://doi.org/10.3358/shokueishi.41.280

ED

Vandenberg, L.N., Colborn, T., Hayes, T.B., Heindel, J.J., Jacobs, D.R., Lee, D.-H., Shioda, T., Soto, A.M., vom Saal, F.S., Welshons, W. V, Zoeller, R.T., Myers, J.P., Myers, J.P., 2012. Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose

PT

responses. Endocr. Rev. 33, 378–455. https://doi.org/10.1210/er.2011-1050 Visvader, J., Stingl, J., 2014. Mammary stem cells and the differentiation hierarchy: current

CC E

status and perspectives. Genes Dev. 28, 1143–1158. https://doi.org/10.1101/gad.242511.114

Wester, P.W., Krajnc, E.I., Leeuwen, F.X.R.V.A.N., Loeber, J.G., Heijden, C.A.V.A.N.D.E.R.,

A

Programme, P.D., 1990. Chronic toxicity and carcinogenicity of bis(tri-n-butyltin)oxide (TBTO) in the rat. Food Chem Toxicol 28, 179–196.

Whalen, M.M., Loganathan, B.G., Kannan, K., 1999. Immunotoxicity of environmentally relevant concentrations of butyltins on human natural killer cells in vitro. Environ. Res. 81, 108–116. https://doi.org/10.1006/enrs.1999.3968 Xu, P., Hong, F., Wang, J., Dai, S., Wang, J., Zhai, Y., 2018. The CAR agonist TCPOBOP

26 inhibits lipogenesis and promotes fi brosis in the mammary gland of adolescent female

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PT

ED

M

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mice. Toxicol. Lett. 290, 29–35. https://doi.org/10.1016/j.toxlet.2018.03.017

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Subtitles

28 Fig. 1. Representative abdominal dissection showing the mammary gland (MG) fat pad in CON and TBT female rats (A, B). CON and TBT 4th MG mounted in glass slides (C, D). Increased MG

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weight in TBT rats (E). (n=7).*p≤0.05 (Student’s t test).

29 Fig. 2. MG whole mount preparation stained with carmine alum in female rats (A-F). MG of TBT rats decreased TEB numbers (arrows, E, G), AB1 score (H), AB2 score (I), lobules core (arrow head, C, F, J) and DF1 (K)compared with MG of CON rats. No significance differences were observed in DF2 (L), MEA (M, O) or elongation (N, P) parameters in CON and TBT rats. (n=4). **p≤0.01, ***p≤0.00 (Student’s t test). TEBs: Terminal end buds. AB1 and 2: Alveolar buds type

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1 and 2. DF1 and 2: Differentiation scores 1 and 2. MEA: Mammary epithelial area.

30 Fig. 3. MG morphology evaluation in female rats using H&E stain. Normal MG morphology aspect, show ing a duct (D) surrounded by adipocytes in CON rats (A, A1). Abnormal MG morphology aspect, showing enlargement of adipocytes, inflammatory cells and connective tissue surrounding MG duct in TBT rats (B, B1). TBT rats had reduced MG adipocytes number (C). TBT rats had a higher MG adipocytes (D). Triglycerides were higher in MG of TBT rats (E). PPARγ protein expression was higher in the TBT rats (F). Mast cells (arrows) stained by Alcian blue in the MG ducts (G, H) and WAT (I, J) of CON and TBT rats. Mast cell number of MG was

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increased in the ducts (K) and WAT(L) of TBT rats. High NAG activity in MG of TBT rats (M). ED1 (N), ERα (O) and CYP19 (P) protein expressions were higher in TBT rats. (n= 4).*p≤0.05,

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****p≤0.0001 (Student’s t test). D: mammary gland duct

Fig. 4. MG oxidative stress assessment in female rats. MG fluorescent micrographs from in situ detection of superoxide anion (O2-) production using O2-sensitive dye DHE (red fluorescence) staining were obtained from CON rats (A) and TBT rats (B). Increased production of O2- was observed in the MG of TBT rats. (C). High TBARS levels were elevated in MG of TBT rats (D).

31 GSH level was decreased in the MG of TBT rats (E). MG GP91-PHOX protein expression in CON and TBT rats (F). Increased MG caspase-3 protein expression in TBT rats (G) (n=4-5).

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*p≤0.05, **p≤0.01 (Student’s t test). D: mammary gland duct

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Fig. 5.MG collagen deposition assessment in female rats. Representative Picro-siruis-stained sections of MG obtained fromf CON (A, A1) rats and TBT (B, B1). Increased collagen deposition was observed in MG of TBT rats (C). Increased MGCOL1A1 and COL1A3 protein expression were observed in TBT rats (D, E). (n=4). *p≤0.05, **p≤0.01 (Student’s t test). D: mammary gland duct

32 Suppl. Fig. 1. (A) Graphical representation of the estrous cycle in the CON and TBT rats by vaginal cytology for 15 days, showing abnormal estrous cyclicity in TBT rats. (B) A graphical representation of each estrous cycle phases duration and the total cycle length. (C) Body weight evaluation during 15 days of treatment in CON and TBT rats. Proestrus (P), estrus (E), metestrusdiestrus (M-D). (n=6-8). *p≤0.05 (Student’s t test). Suppl. Fig. 2.TBT rats had lower ovarian (A)and uterine(B)weights. MPO activity in MG ofCON and TBT rats(C). (n=5-6). *p≤0.05 (Student’s t test for MG reproductive tract weight and MPO

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activity).

Suppl. Fig. 3. Correlation between the MG adipogenic markers, inflammation, oxidative stress,

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fibrosis, CYP19 and ERα protein expression. The values of MG inflammation (A), oxidative stress (B) and fibrosis (C) were correlated with MG fat pad adipocytes diameter. MG

inflammation (D), oxidative stress (E) and fibrosis (F) were correlated with MG TG levels. MG inflammation (G), oxidative stress (H) and fibrosis (I) were correlated with MG PPARγ protein

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expression. The values of MG inflammation (J), oxidative stress (K) and fibrosis (L) were correlated with MGCYP19 protein expression. MG inflammation (M), oxidative stress (N) and

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fibrosis (O) were correlated with MG ERαprotein expression (n=8). Statistical significance (p ≤

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distribution, respectively, was detected.

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33 Table 1. Summary of changes to the mammary gland induced by OTs. Model/OT/Dose/ Time of exposure MCF-7, MDA-MB-231, MTSV-17/ OTcs (0.00110µM)/48-72hrs

MCF-7/TBT (0-1000 nM)/ 20-24hrs

MCF-7/ TBT, TPT/(10-12106M)/24-72hrs

MCF-7, MDA-MB231/ TBT, TPT/(200800 nM)/72 hrs

Rats/ TBT/ 100ng/Kg/day / 15 days

NR NR NR NR

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

↑ ↓ ↓ ↓

NR NR ↑ NR

NR NR NR NR

NR NR NR NR

NR NR NR NR

NR NR NR NR

↓/↑ ↑ NR ↑

NR

NA

NA

NA

NA

↑

NR

NA

NA

NA

NA

↑

NR NR NR

NR NR NR

NR NR NR

NR NR NR

NR NR NR

↑ ↑ ↓

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Mice/TBT(0.5 mg/kg/day)/ GD 12-21

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Mammary gland parameters Morphology MG weight TEBs Lobules Differentiation (ABs, DF1) Lipid metabolism Adipocytes (Nº /diameter) MG triglycerides Lipid deposition (Oil Red) PPARγ expression Inflammation Mast cells number Macrophage (activity/expression) Oxidative Stress DHE TBARS GSH Fibrosis Collagen deposition COL1/3 expression

NA NA ↑ NA NA ↑ Fickova; Grünet al, Sharan et al, Balaset al, 2011, 2012 Macho; Brtko, Hunakovaet al, 2015 Reference This study 2006 2013 2014 MG: Mammary gland; GD: gestational day. TEBs: Terminal end buds; ABs: Alveolar buds; DF1: Score of differentiation 1; PPARγ: Peroxisome proliferatoractiveted receptor gamma; DHE: Dihydroethidium assay; TBARS: Thiobarbituric acid reactive substances; GSH: Reduced glutathione; COL1/3: Collagen types 1 and 3; OTs: Organotins; TBT: Tributyltin; TPT: Triphenyltin; DPT: Diphenyltin; OTcs: TBT, TPT, DPT complexes; MCF-7: breast, estrogen receptor ERpositivecells;MDA-MB-231:breast, ER negative cells;MTSV-17:normal immortalized human mammary gland epithelial cell;↑: Increased; ↓: Decreased; ↔: Unchanged or similar to control; NR: Not reported; NA: Not applicable.hrs: hours. Nº: number. NA NA

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Table 2 - Correlation between the MG adipogenic markers, morphology/development, CYP19 and ERα protein exp MG morphology/development and reproductive markers TEB AB1 AB2 Adipogenic markers

Lobule

DF1

Pearson r

P

Pearson r

P

Pearson r

P

Pearson r

P

Pearson r

P

WA diameter

-0.5042

0.202

-0.5192

0.187

-0.6983

0.054

-0.5377

0.169

-0.6558

0.077

TG

-0.8776

0.004

-.8167

0.013

-0.5364

0.170

-0.7622

0.027

-0.8077

0.015

PPARγ

-0.3906

0.338

-0.8535

0.007

-0.2047

0.626

-0.2792

0.503

-0.5437

0.163

CYP19

-0.4554

0.256

-0.7166

0.045

-0.6955

0.054

-0.5650

0.144

-0.7517

0.031

ERα

-0.3747

0.360

0.5923

0.121

-0.5287

0.178

-0.3212

0.438

-0.5551

0.153

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Reproductive markers

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MG: Mammary gland; TEB: Terminal end bud; AB1: Alveolar bud; type 1; AB2: Alveolar bud type 2; DF1: Score of differentiation 1; CY19: MG aromatase protein expression; ER: MG estrogen receptor  protein expression; WA: White adipocyte; TG: MG triglycerides levels; PPARγ: MG peroxisome proliferator-activated receptor gamma protein expression.