Subacute and low dose of tributyltin exposure leads to brown adipose abnormalities in male rats

Abstract

Tributyltin (TBT) is an obesogenic endocrine disrupting chemical (EDC) linked with several metabolic complications. Brown adipose tissue (BAT) is the principal site for thermogenesis, making it a potential target for obesity management and metabolic disease. However, few studies have evaluated TBT effect on BAT function. In this investigation, we assessed whether subacute (15 days) and low dose of TBT exposure (100 ng/kg/day) results in abnormal BAT morphophysiology in adult male rats. Body temperature, BAT morphology, inflammation, oxidative stress, collagen deposition and BAT metabolic gene expression markers were assessed in room temperature (Room, ~24 °C) and after cold tolerance test (Cold, ~4 °C) conditions. A reduction in body temperature was observed in both Room and Cold conditions in TBT rats, suggesting abnormal BAT thermogenic function. Changes in BAT morphology were observed in TBT rats, with an increase in BAT lipid accumulation, an increase in BAT unilocular adipocyte number and a decrease in BAT multilocular adipocyte number in Room condition. All these parameters were opposite in Cold condition TBT rats, leading to a borderline increase in BAT UCP1 protein expression. An increase in BAT mast cell number was observed in TBT rats in Room condition. An increase in ED1 protein expression (macrophage marker) was observed in TBT rats in Cold condition. Oxidative stress and collagen deposition increased in both Room and Cold conditions in TBT rats. TBT exposure caused a borderline increase in BAT COL1A1 protein expression in Cold condition. Further, strong negative correlations were observed between body temperature and BAT lipid accumulation, and BAT lipid accumulation and multilocular adipocyte number. Thus, these data suggest that TBT exposure impaired BAT morphophysiology through impacts on lipid accumulation, inflammation, fibrosis and oxidative stress in male rats.

Graphical Abstract

1. Introduction

Tributyltin (TBT) is a persistent organotin contaminant that is widely used in several agroindustry applications, such as manufacturing of antifouling paints, as a preservative in woods, papers, and textiles, in broad-spectrum biocides, and as a stabilizer in plastic production (Fent, 1996; Barbosa et al., 2021). Exposure to the biocide TBT remains a significant public health concern because of its bioaccumulative properties and a long history of widespread global use (Antizar-Ladislao, 2008; Barbosa et al., 2021; US EPA IRIS, 1997). Worldwide production of TBT compounds was estimated at about 5000 ton/year and production of organotin compounds was approximately 50,000 ton/year in 1996 (Alzieu, 1998; EEA, 2001). Despite the efforts of the International Marine Organization, through the International Convention on the Control of Harmful Antifouling Systems on Ships, to ban the use of TBT-based antifouling paints (September 2008) and the Rotterdam Convention to forbid the trade of tributyltin, antifouling paints containing TBT as an active ingredient are still being registered for commercialization today. In fact, recent studies have reported that TBT may have even greater global distribution than previously thought (Paz-Villarraga et al., 2015; Uc-Peraza et al., 2022).

TBT is detected in sea sediment (0.5–20,000 ng/g), seawater (0.1–281,8 ng/l), and seafood (0.15–19,757 ng/g), representing an environmental issue along coastal areas under the influence of maritime activities, such as Europe (Furdek et al., 2012), Asia (Garg et al., 2011), South Africa (van Gessellen et al., 2018), Oceania (Roach e Wilson, 2009), North America (Keithly et al., 1999; Tallmon, 2012), and South America (Abreu et al., 2021; Castro et al., 2018; Batista-Andrade et al., 2018). The European Food Safety Authority and the United States Environmental Protection Agency have established human tolerable daily intake of 250 and 300 ng/kg/day of TBT, respectively. These values were derived by applying a 100-fold safety factor from the no-observed-adverse-effect level of 25 μg/kg/d (ESFA, 2004; US EPA IRIS, 1997; Vos et al., 1990). TBT (155 ng/ml) has been detected in human blood from study participants in Michigan, USA (Whalen et al., 1999). Other studies have shown that TBT levels in human tissue range between 0.01 and 85.0 ng/g (Kannan et al., 1996; Nielsen e Strand, 2002; Rantakokko et al., 2013, 2014).

Exposures to obesogenic environmental endocrine disrupting chemicals (EDCs) may play a significant role in the worldwide obesity epidemic. TBT is a representative obesogen and a potent EDC that activates nuclear hormone receptors, including the retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ), which together form a heterodimer to regulate adipocyte differentiation (Chamorro-García et al., 2013; Grün e Blumberg, 2006). TBT exposure is associated with several metabolic disfunctions, such as obesity, inflammation, and oxidative stress in fat, liver, pancreas, and thyroid and abnormal adipogenic genes expression in fat and liver of animal models (Andrade et al., 2018; Freitas-Lima et al., 2018; Chamorro-García et al., 2013; Grün e Blumberg, 2006). Our previous studies in female rats reported that a subacute and low dose of TBT (100 ng/kg/day for 15 days) led to obesity, hyperleptinemia, dyslipidemic lipid profile, insulin resistance, reduction in islet pancreatic number, presence of lipid droplets and granuloma inside of liver, abnormal thyroid morphophysiology and presence of inflammation, oxidative stress and collagen deposition in white adipose tissue (WAT) (Bertuloso et al., 2015; Freitas-Lima et al., 2018; Rodrigues-Pereira et al., 2022)

Recent human and rodent investigations have reported that brown adipose tissue (BAT) is emerging as a therapeutic target for metabolic disease such as obesity (Saito et al., 2009; Rangel-Azevedo et al., 2022). BAT is responsible for the dissipation of chemical energy to heat, known as thermogenesis (Orava et al., 2011; Saito et al., 2009; Wicksteed e Dickson, 2017). Decreased energy expenditure and body temperature are strongly associated with increased susceptibility for obesity, suggesting an important role for BAT in body metabolic rate (Chiang et al., 2009; Klaus et al., 1998; Landsberg, 2012; Matsushita et al., 2014; Ouellet et al., 2011). Recent studies have observed that obesity is associated with BAT dysfunction, such as changes BAT morphology, leading to whitening of brown adipocytes and impaired thermogenesis (Rangel-Azevedo et al. 2022). Importantly, proper BAT function relies on hormone receptors and signaling pathways that make BAT susceptible to disruption by EDCs, such as TBT. Since the discovery that TBT is an EDC, few studies have reported its effect on BAT models in vivo and in vitro (Chamorro-García et al., 2013; Shoucri et al., 2018). Chamorro-García et al., 2013 observed a modest effect in BAT morphology in adult male mice and a more modest or unchanged effect in female mice after prenatal TBT exposure.

Thus, the effects of TBT on BAT function remain unclear. In the present study, we hypothesized that our dosing model using a subacute and low dose of TBT would lead to BAT abnormalities such as abnormal thermogenesis, cold intolerance, BAT lipid accumulation, inflammation, oxidative stress, and fibrosis via an endocrine disruption mechanism.

2. Methods

2.1. Chemicals

Tributyltin chloride (TBT, 96%, Sigma, St. Louis, Mo., USA) was dissolved in 0.4% ethanol following protocols from previous studies performed in our laboratory (B.D. Bertuloso et al., 2015; Ceotto Freitas-Lima et al., 2018).

2.2. Experimental animals

Adult male Wistar rats (12 weeks old) were maintained under controlled temperature between 24 ± 1°C (room temperature, referred to as Room condition) with a 12:12 h light/dark cycle. Rat chow and filtered tap water were provided at libitum. All protocols were approved by the Ethics Committee of Animals of the Federal University of Espirito Santo (UFES) (60/2017). The rats were divided into two groups: control (CON, n=10) rats were treated daily with vehicle (0.4% ethanol) and TBT (TBT, n=10) rats were treated daily with TBT (100 ng/kg/day) for 15 days by gavage. All animals were anesthetized using ketamine and xylazine (90 mg/kg and 4.5 mg/kg, intraperitoneal injection) on the last day of treatment (day 15) and euthanized by decapitation. Blood samples were obtained for future biochemical analysis and wet organs were dissected, isolated, and weighed at Room condition (Suppl. Table 1, n=5). Oral exposure to TBT in male rats was chosen so we could compare the current findings with our previous work demonstrating that TBT exposure causes metabolic complications (Bertuloso et al. 2015; Sena et al. 2017). Further, the principal route of exposure to TBT is ingestion by consumption of contaminated food. In addition, the TBT dose used in the current study (100 ng/kg) was approximately 3 times lower than the tolerable daily intake level of 300 ng/kg for humans established by the U.S. Environmental Protection Agency (USEPA, 1997) and presents a similar concentration range as found in human tissue (Kannan et al., 1996; Nielsen e Strand, 2002; Rantakokko et al., 2013, 2014).

2.3. Adiposity and lipid profile

To evaluate metabolic parameters of CON and TBT rats, we assessed body weight, adiposity (the sum of the weights of the parametrial, abdominal, retroperitoneal, and perirenal fat adipose tissues), liver weight, and morphology at Room condition (Suppl. Table S1) and after cold tolerance test condition (Cold, Suppl. Table S3), described in detail below (Bertuloso et al., 2015). Serum cholesterol total (CT), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride (TG) levels were evaluated using colorimetric kits according to the manufacturer’s directions at Room (Suppl. Table S2) and Cold condition (Suppl. Table S3) (Bioclin) (Bertuloso et al., 2015; Freitas-Lima et al., 2018).

2.4. Glucose tolerance and insulin sensitivity analysis

An insulin sensitivity test (IST) was performed without fasting (Suppl. Fig. 2). The animals received insulin via intraperitoneal injection using sterile saline solution (n=5, 0.75 U/kg body weight, Sigma-Aldrich). The tail blood glucose levels were recorded at 0, 30, 60, 90 and 120 min after insulin injection. To evaluate the glucose tolerance test (GTT) (Suppl. Fig. 2), fasted rats received D-glucose (2 mg/g of body weight) via intraperitoneal injection using sterile water and tail blood glucose levels were recorded at 0, 15, 30, 60, and 90 min using an Accu-Chek glucometer in other set of rats (n=5) (Roche Diagnostics Corp, Ind) (Bertuloso et al., 2015; Freitas-Lima et al., 2018).

2.5. Hormonal assays

Serum insulin, adiponectin, leptin, free T4 and total T3 levels were measured via enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Diagnostic Prod. Corporation, LA, CA) at Room (Suppl. Fig. 1, 2 and 4) and Cold condition (Suppl. Table 4) (Freitas-Lima et al., 2018; Kuriyama et al., 2007).

2.6. Hepatic enzymes activities assessment

Serum glutamic pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) activities were measured using colorimetric kits according to the manufacturer’s directions at Room (Suppl. Fig. 3) and Cold condition (Suppl. Table 4) (Bioclin, Belo Horizonte, MG, Brazil) (Bertuloso et al., 2015).

2.7. Morphological analysis

The epididymal adipose tissue (EAT) (Suppl. Fig.1), pancreas (Suppl. Fig.2), liver (Suppl. Fig. 3) and thyroid gland (Suppl. Fig.4) were removed from CON and TBT rats from Room condition and fixed in PBS-formalin (4%) (pH 7.4) for 24 – 48 hours at room temperature. In addition, BAT tissues were removed from CON and TBT rats at Room and Cold condition and fixed as described above. Paraffin-embedded organs were sectioned into 5-μ-mthick slices and stained with hematoxylin and eosin (H&E); the slices were examined as high-quality images (2048×1536 pixels) using an Olympus microscope (AX70; Olympus, Center Valley, PA), photographed with an AxioCamICc1 camera and exported to AxioVision Software (4.8 software). Photomicrographs were obtained using a 10 and 100x objectives and were used to assess the general organizational structure and cellular composition (Bertuloso et al., 2015; Sena et al., 2017). The EAT adipocyte diameter was determined as the mean of distance into the major and minor adipocyte diameters (Ludgero-Correia et al., 2012). The pancreatic islets number was counted and represented as islet number per square millimeter (Yamabe et al., 2010). The thyroid follicle number was counted and represented as follicle number per square millimeter and the follicle diameter was determined as the mean distance between the major and minor follicles (Rodrigues-Pereira et al., 2022). BAT lipid content is shown as the percentage of the area covered by lipid vesicles, measured using ImageJ. The number of BAT blood vessels was counted and represented as blood vessel number per square millimeter (Chamorro-García et al., 2013; Schneider et al., 2012; Shimizu et al., 2014).

2.8. Measurement of rectal temperature and cold tolerance test

The animals were kept at 24 ± 1°C (room temperature, Room condition, n=10 for each group). We evaluated if our handling procedure contributed to rectal body temperature changes during the acclimation period. We performed this evaluation 5 days before (day −5) to start the experiment (day 0). We did not observe significant body temperature changes in future TBT rats on day −5 (33.78 ± 0.08 °C) and day 0 (33.76 ± 0.04 °C) compared to control rats (day −5: 33.80 ± 0.13 °C; day 0: 33.81 ± 0.06 °C), suggesting our handling procedure is appropriate. In addition, on measurement days, the animals had a habituation time of 2 hours to the test environment. The animals were immobilized before the test, having undergone adaptation to the immobilization previously. Baseline measurements of rectal body temperatures were performed with a rectal thermometer (Physitemp, RET-3 probe). The entire handling procedure was performed by a researcher who was properly trained in this protocol, avoiding any discomfort to the animal and ensuring adequate data collection (Merlo et al. 2019; Merlo et al. 2016). To obtain a rectal temperature, the animal is hand-restrained and placed on a horizontal surface, e.g., a cage lid. The tail is then lifted, and a probe is gently inserted into the rectum to a fixed depth (typically, up to 2 cm) (Meyer et al., 2017).

Cold tolerance test (Cold condition or CTT) was carried out in temperature-controlled rodent incubators. The rats were placed in pre-cooled cages at 4–5°C with bedding, wood shavings, and free access to standard food and water (n=10 for each group). Body temperature was measured every 60 min for a period of 6 h. Animals with a temperature reduction below 30 °C were removed from the protocol (Emmett et al., 2017; Meyer et al., 2017). We also evaluated BAT morphophysiology and gene expression after Cold condition. Thus, BAT parameters were evaluated in control and TBT rats exposed at room temperature (Room) and cold condition (Cold), using different sets of animals.

2.9. Inflammation assessment

All tissue sections from Room (BAT, EAT, pancreas, liver, thyroid, Fig. 3, Suppl. Fig. 1, 2, 3 and 4) and Cold conditions (BAT, Fig. 3) were stained with Alcian Blue according to a standard protocol (Sigma-Aldrich Co, LLC). Each of the 5-μm sections was used to obtain photomicrographs (10 and 100x objectives). The number of positively stained cells (i.e., cells containing purple cytoplasmic granules) within the tissue was evaluated. The number of positively stained cells was then expressed per unit area (mm²) (Bertuloso et al., 2015; Merlo et al., 2016). Neutrophils and macrophages present were indirectly measured by myeloperoxidase (MPO) and n-acetyl-β-D-glucosaminidase activity (NAG), which are present at high levels in activated neutrophils and macrophages, respectively (Araújo et al., 2010; Barcelos et al., 2005).

Fig. 3.

BAT inflammation evaluation in male rats. Representative Alcian Blue stained in BAT from CON (A, E) and TBT (B, F) from room condition, and CON (C, G) and TBT (D, H) after CTT (cold condition). (I) BAT mast cell number. (J) BAT MPO activity showing neutrophil presence. (K) BAT NAG activity showing macrophage presence. (L) ED1 and (M) IL-6 protein expression. Means with the same letter are not significantly different from each other; a, b, c and d: p < 0.05. Two-way ANOVA (Tukey’s multiple comparison test), n=5–6. Arrowhead: mast cell; BAT: brown adipose tissue; MPO: myeloperoxidase; NAG: n-Acetyl-β-D-Glucosaminidase. CTT: cold tolerance test.

2.10. Collagen deposition assessment

BAT sections from Room and Cold conditions were stained with Picrosirius Red and Masson’s trichrome (TM, Suppl. Fig. 1) to obtain photomicrographs with a 10x objective under phase contrast and analyzed using ImageJ. The images were converted into high contrast black and white images to visualize stained collagen fibers. The results represent the percentage of collagen deposited in the BAT (Merlo et al., 2016).

2.11. Oxidative stress assessment

BAT cryosections Room and Cold conditions were obtained by Leica cryostat (7 μm, n = 5) and incubated with the superoxide anion (O₂⁻) sensitive fluorescent dye dihydroethidium (DHE) (Merlo et al., 2018). Sections were mounted with Vectashield (H-1000; Vector Laboratories). Images were acquired using an inverted fluorescence microscope (Leica DMi8) equipped with a charge-coupled device digital camera (Leica dfc365 fx) for image capture and processing with LAS X LS software. Images were captured using fluorescence filter FITC/TRITC detection (red). The signal intensity was analyzed in 20 sections. The images were acquired at the LHMI Laboratory, UFES, Brazil

2.12. Immunoblotting assessment

BAT protein levels were obtained from Room and Cold condition samples (da Costa et al., 2019). Briefly, protein samples (n = 4–5) were loaded onto an SDS/PAGE gel to perform the immunoblotting analysis (Bio-Rad). The primary antibodies included anti-uncoupling protein 1 (UCP1, #sc-293418, 1:500, SCBT, INC), anti-peroxisome proliferative activated receptor, gamma, coactivator 1 (Pparg1a or PGC1α, #M00236, Boster Biological Technology, CA), anti-vascular endothelial growth factor receptor (VEGFr, #PA5–16487, Themo Fisher Scientific, US), anti-ED1 (MCA341GA, 1:500, Bio-Rad, INC), anti-interleukin 6 –(IL-6, #sc-57315, 1:500, SCBT, INC), anti-collagen, type 1, alpha 1 (COL1A1, #sc-293182, 1:500, SCBT, INC) and anti-GAPDH (sc25778, 1:1000, SCBT, INC). UCP1, VEGFr, ED1, IL-6 and COL1A1 proteins were detected using a secondary anti-mouse IgG alkaline phosphatase conjugate (A3562, 1:1000, Sigma). PGC1α and GAPDH protein were detected using a secondary anti-rabbit IgG alkaline phosphatase conjugate (A3687, 1:1000, Sigma). The blots for UCP1, VEGFr, ED1, IL-6, COL1A1 and their respective GAPDH control were visualized using a color development reaction containing BCIP/NBT solution (sc24981, SCBT, INC). The protein bands were analyzed by densitometry using ImageJ software. The relative expression levels were normalized by dividing the values of the protein of interest by the corresponding internal control values. The total protein level measurement was performed at the LABIOM Laboratory, UFES, Brazil.

2.13. Statistical analysis

All data are reported as the mean ± SEM. To identify possible outliers in the data, a two-sided Grubbs’ test was used. When the 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. Comparisons between the groups were performed using Student’s t-tests for Gaussian data. Additionally, for the non-Gaussian data, a Mann-Whitney test was used. Two-way ANOVA was used for graph lines to verify the interaction between the independent variables (time and strain) and was followed by Bonferroni post-test. A value of p < 0.05 was regarded as statistically significant. When p-values were > 0.05, but < 0.10, data were considered to exhibit borderline significance. 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. Statistical analyses and graphical construction were performed using GraphPad Prism version 6.00 (La Jolla, CA, USA).

3. Results

3.1. TBT exposure effects in body temperature

TBT rats presented a reduction in rectal temperature at day 15 compared with CON rats in room temperature conditions (Room, ~24 °C) (Fig. 1A, p < 0.05). A reduction in rectal temperature at 1 and 4 hours and 0.8% decrease of area under the curve (AUC) during the cold tolerance test (Cold, ~4 °C) were observed in TBT rats compared with CON rats (Fig. 1B and C, respectively, p < 0.05).

Fig. 1.

Rectal temperature in male rats. (A) Rectal temperature in room temperature. (B) Rectal temperature during CTT in male CON and TBT rats. (C) AUC of CTT. *p < 0.05 vs CON. (D) UCP1 and (E) PGC1α protein expression at Room condition. (F) UCP1 and (G) PGC1α protein expression at Cold condition. Two-way ANOVA (Tukey’s multiple comparison test in A and B). (Student’s t-test), n=10. AUC: area under the curve; CTT: cold tolerance test (cold condition).

3.2. TBT exposure and BAT UCP1 and PGC1α protein expression

No significantly changes were observed in BAT UCP1 and PGC1α protein expression between CON and TBT rats at Room condition (Fig. 1D and E, p > 0.05 and p > 0.05). A tendency toward increased BAT UCP1 protein expression was observed in TBT rats compared with CON rats after Cold condition (Cold, ~50%, p = 0.06, Fig. 1F). Despite a reduction of approximately 50%, TBT exposure did not significantly reduce PGC1α protein expression compared to CON after Cold condition (Fig. 1G, p > 0.05).

3.3. TBT exposure and other metabolic evaluation

Body weight, adiposity, epididymal adipose tissue (EAT) morphology, mast cell number, adipokine levels were similar between CON and TBT rats in Room condition (Suppl. Fig. 2, p > 0.05). Similarly, no changes were observed in wet organs between CON and TBT rats at Room condition (Suppl. Table S1, p > 0.05). In the lipid profile, we only observed an increase in serum LDL levels in TBT rats compared to CON rats (Suppl. Table S2, p < 0.05). No changes in glucose metabolism and pancreas morphology were observed in CON and TBT rats at Room condition (Suppl. Fig. 3). In the liver, we only observed a reduction in mast cell number in TBT rats compared to control at Room (Supp. Fig. 4, p < 0.05). An increase in thyroid follicles and a reduction serum free T4 levels in were observed in TBT rats compared to CON rats at Room condition (p < 0.05, Suppl Fig. 5).

Body weight, adiposity, and fat deposit weights were similar between CON and TBT rats after Cold condition (Suppl. Table S3, p > 0.05). Similarly, no changes were observed in lipid profile, hepatic enzymes, serum insulin, adiponectin, leptin, T4 and T3 levels between CON and TBT rats after Cold condition (Suppl. Table S4).

3.4. TBT exposure induces BAT morphophysiology abnormalities

BAT H&E stained sections from CON rats contained regular morphology, with multilocular adipocyte and small lipid droplets in Room condition (~24 °C) (Fig. 2A). However, the BAT of TBT rats presented a small increase in unilocular adipocyte number (Fig. 2B) that contributed to an increase in lipid accumulation compared to CON rats in Room condition (8%, p < 0.05, Fig. 2E). Both CON and TBT rats showed a reduction in BAT lipid accumulation in Cold condition (~4 °C) compared to respective CON and TBT rats from Room condition (11% and 39%, respectively, p <0.05, Fig.2E). TBT rats presented a reduced number of lipid droplets compared to CON rats in Room condition (10%, p < 0.05, Fig. 2F). However, TBT rats presented an increased number of lipid droplets compared to CON rats in Cold condition (15%, p < 0.05, Fig.2F). TBT rats presented an increase in number of unilocular adipocytes and a reduction of multilocular adipocyte, compared to CON rats in Room condition (11% and 8%, respectively, p < 0.05, Fig.2G and H). However, TBT rats presented a reduced number of unilocular adipocytes and an increase in multilocular adipocytes compared to CON rats in Cold condition (15% and 28%, respectively, p < 0.05, Fig.2G and H).

Fig. 2.

BAT morphology in male rats. Representative H&E-stained BAT CON (A) and TBT (B) in room temperature condition (room), and CON (C) and TBT (D) in cold condition after CTT. (E) Lipid accumulation in BAT. (F) Lipid droplets number in BAT. (G) Unilocular adipocytes number in BAT. (H) Multilocular adipocytes number in BAT. (I) Blood vessels number in BAT. (J) VEGFr protein expression. Means with the same letter are not significantly different from each other. Two-way ANOVA (Tukey’s multiple comparison test), n=5–6. Arrowhead: blood vessels.

3.4. BAT blood vessel number

TBT rats showed no significant changes in BAT blood vessel number compared to CON rats in Room condition (p > 0.05, Fig.2I). In Cold condition, BAT blood vessel number was increased in CON rats, whereas BAT blood vessel number in TBT rats remained the same as room temperature conditions (p < 0.05, Fig.2I).

3.5. TBT exposure and BAT VEGFr protein expression

No significantly changes were observed in BAT VEGFr protein expression between CON and TBT rats at Room and Cold conditions (p < 0.05 and p < 0.05, Fig.2J).

3.6. TBT exposure increased mast cell number

An increase in BAT mast cell number was observed in the TBT rats compared to the CON rats in Room condition (157%, p < 0.05, Fig. 3A, B, E, F, I). No significant changes were observed in BAT mast cell number between CON and TBT rats in cold conditions (p > 0.05, Fig. 3C, D, G, H and I). In addition, CON rats showed an increase in BAT mast cell number in Cold conditions compared to CON rats from room temperature conditions (300%, p < 0.05, Fig. 3I).

The BAT MPO and NAG activities, which indicate neutrophil and macrophage presence, were similar between CON and TBT rats in both Room and Cold conditions (p < 0.05 and p <0.05, Fig. 3J and K).

3.7. TBT exposure and BAT ED1 and IL-6 protein expression

No statistically significant changes were observed in BAT ED1 protein expression (macrophage marker) between CON and TBT rats at Room condition (Fig. 3L, p > 0.05). An increase in BAT ED1 protein expression was observed in TBT rats compared to CON rats after Cold condition (Fig. 3M, p < 0.05). No significantly changes were observed in BAT IL-6 protein expression between CON and TBT rats at Room and Cold condition (p < 0.05 and p < 0.05, Fig.3N).

3.8. TBT exposure induces BAT collagen deposition

An increase in BAT collagen deposition (performed with picrosirius staining) was observed in the TBT rats compared with the CON rats at Room condition (~24 °C) (25%, p < 0.05, Fig. 4A, B and E). Similarly, high BAT collagen accumulation was observed in TBT rats compared to CON rats in Cold condition (~4 °C) (22%, p < 0.05, Fig. 4C, D and E). Both CON and TBT rats showed increased BAT collagen deposition in Cold condition compared to respective CON and TBT rats in Room condition (p < 0.05, Fig. 4A, C, B, D and E). The same evaluation was performed using Masson’s trichrome staining. An increase in BAT collagen deposition was observed in the TBT rats compared with the CON rats at Room condition (39%, p < 0.05, Suppl. Fig. 1). Despite an increase of approximately 18%, TBT exposure did not significantly increase collagen deposition compared to control in Cold condition (p > 0.05, Suppl. Fig. 1).

Fig. 4.

BAT collagen deposition evaluation in male rats. Representative Picrosirius staining in BAT of CON (A) and TBT (B) rats from room temperature condition, and CON (C) and TBT (D) rats from cold condition. (E) BAT collagen deposition. (F) COL1A protein expression. Means with the same letter are not significantly different from each other; a, b, and c: p < 0.05. Two-way ANOVA (Tukey’s multiple comparison test), n=5–6.

3.9. TBT exposure and BAT COL1A1 protein expression

No statistically significant changes were observed in BAT COL1A1 protein expression between CON and TBT rats at Room condition (Fig. 4F, p > 0.05). TBT exposure caused a borderline increase in BAT COL1A1 protein expression in TBT rats compared to CON rats after Cold condition (Fig. 4F, p = 0.06).

3.10. TBT exposure induces BAT oxidative stress

BAT oxidative stress was assessed using the fluorescent dye DHE to evaluate superoxide anion levels. An increase in BAT superoxide anion levels was observed in the TBT rats compared with the CON rats in Room condition (17%, p < 0.05, Fig. 5A, B and E). Similarly, a high BAT superoxide anion levels was observed in TBT rats compared to CON rats in Cold condition (6%, p < 0.05, Fig. 5C, D and E). No significant differences were observed between rats of the same treatment group across both temperature conditions (p > 0.05, Fig. 5E)

Fig. 5.

BAT oxidative stress evaluation in male rats. Representative DHE fluorescence in BAT of CON (A) and TBT (B) rats from room temperature condition, and CON (C) and TBT (D) rats from cold condition. (E) BAT DHE fluorescence. Means with the same letter are not significantly different from each other; a and b: p < 0.05. Two-way ANOVA (Tukey’s multiple comparison test), n=5–6.

3.11. Correlation among body temperature, T4 levels, BAT parameters (lipid accumulation, unilocular and multilocular adipocyte, mast cell number, oxidative stress)

To evaluate the relationship between the body temperature (rectal temperature), BAT parameters (i.e., lipid accumulation, unilocular and multilocular adipocyte numbers, inflammation-mast cell numbers and superoxide anion levels) and serum free T4 levels, pairwise correlation analyses were performed, and a linear fit was plotted.

Several BAT markers and abnormalities were correlated in room temperature and cold conditions. We observed a trend towards a negative linear correlation between body temperature and BAT lipid accumulation at room temperature (Fig. 6A, p = 0.0688). However, no significant linear correlation was observed between body temperature and BAT lipid accumulation in cold conditions (Fig. 6B, p = 0.3224).

Fig. 6.

The correlation among rectal temperature, serum free T4 levels and BAT parameters (lipid accumulation, multilocular adipocytes number, mast cell number and oxidative stress). The values of rectal temperature from both room (A) and cold temperature condition (B), were plotted with the BAT lipid accumulation. The values of BAT lipid accumulation from both room (C) and cold temperature condition (D) were plotted with the serum T4 levels. The values of BAT lipid accumulation from both room (E) and cold temperature condition (F) were plotted with the BAT multilocular adipocytes number. The values of BAT lipid accumulation from both room (G) and cold temperature condition (H) were plotted with the BAT mast cell number. The values of BAT multilocular adipocytes number from both room (I) and cold temperature condition (J) were plotted with the BAT mast cell number. The values of BAT DHE fluorescence (oxidative stress) from both room (K, M) and cold temperature condition (L, N) were plotted with the BAT lipid accumulation and BAT mast cell number, respectively. Statistical significance (p < 0.05) was tested using the Spearman’s or Pearson’s test if a non-Gaussian or Gaussian data distribution, respectively, was detected.

A trend towards a negative linear correlation was observed between the BAT lipid accumulation and T4 levels in room temperature conditions (Fig. 6C, p = 0.0755). Interestingly, a positive linear correlation was observed between T4 levels and BAT lipids accumulation in cold conditions (Fig. 6D, p < 0.05). A negative linear correlation was observed between room temperature condition stress and COL1A1 protein expression (data not shown, p = 0.0167). BAT lipid accumulation was negatively correlated with BAT multilocular adipocyte number in both room temperature and cold conditions (Fig. 6E, p < 0.05 and Fig. 6F, p <0.05).

BAT lipid accumulation was positively correlated with BAT mast cell number in both room temperature and cold conditions (Fig. 6G, p < 0.001 and Fig. 6H, p <0.05). BAT multilocular adipocyte number was negatively correlated with mast cell number in room temperature conditions (Fig. 6I, p < 0.05). However, no significant correlation was observed between BAT multilocular adipocyte number and mast cell number in cold conditions (Fig. 6J, p = 0.3225).

BAT oxidative stress was positively correlated with BAT lipid accumulation at room temperature (Fig. 6K p < 0.001). However, BAT oxidative stress was negatively correlated with BAT lipid accumulation in cold conditions (Fig. 6L, p < 0.05). A positive linear correlation was observed between BAT oxidative stress and BAT mast cell number at room temperature (Fig. 6M, p < 0.001). No significant correlation was observed between BAT oxidative stress and mast cell number in cold conditions (Fig. 6N, p = 0.7290).

4. Discussion

Our study provides evidence that subacute low dose TBT exposure for 15 days is responsible for reduction in body temperature in both room temperature and cold tolerance test conditions in male rats. TBT exposure alters BAT morphology involved lipid accumulation, lipid droplet remodeling, inflammation, fibrosis, and oxidative stress. Moreover, our results show that TBT exposure changes BAT metabolic, inflammatory and collagen maker expression (UCP1, ED1 and COL1A) in cold condition. Collectively, these data indicate that TBT exposure alters BAT morphology and gene expression that contribute to abnormal thermogenesis control in male rats (Table 1).

Table 1:

Summary of BAT changes induced by TBT exposure

Parameters	BAT condition
	Room temperature	Cold tolerance test
BAT function
Body temperature	↓	↓
Mitochondria number	↓	NE
Mitochondria cristae	↓	NE
UCP1expression	↔	b↑
PGC1α expression	↔	↔
Lipid and blood vessel evaluation
Lipid accumulation	↑	↓
Unilocular adipocyte	↑	↓
Multilocular adipocyte	↓	↑
Blood vessel number	↔	↔
VEGFr expression	↔	↔
Inflammation and ROS evaluation
Mast cell presence	↑	↔
ED1 expression	↔	↑
Il6 expression	↔	↔
Superoxide anion	↑	↑
Collagen deposition evaluation
Picrosirius red staining	↑	↑
Masson’s Trichrome staining	↑	↔
Col1A1 expression	↔	b↑

TBT: Tributyltin chloride. ROS: reactive oxygen species. ↑: increased. decreased. ↔: unchanged or similar to control. b: borderline. NE: not evaluated

TBT is an obesogenic chemical that is associated with adverse metabolic consequences, which can be inherited transgenerationally in both sexes (Chamorro-García et al., 2013). Our previous studies reported that 100 ng/kg/day TBT for 15 days lead to several metabolic abnormalities, such obesity, white adipocyte expansion, insulin resistance (IR), hyperleptinemia, hypoadiponectinemia, dyslipidemic lipid profile, such as high LDL levels, irregular liver, pancreas and thyroid function, and white adipose tissue inflammation in female rats (Bertuloso et al., 2015; Freitas-Lima et al., 2018; Rodrigues-Pereira et al., 2022; Sena et al., 2017). Si et al. (2011) showed that 5 μg/kg TBT for 45 days caused body weight gain and an increase in testicular fat deposit in male mice (Si et al., 2011). In our current study in male rats, no significant changes were observed in body weight/adiposity, epididymal fat, liver, and pancreas function. We observed an increase in serum LDL levels and thyroid follicle numbers and a reduction in T4 levels. Similar results have been reported by Rodrigues-Pereira et al. 2022, 2020. Penza et al. (2011) showed that a 60-day exposure to TBT (0.5 μg/kg) caused an increase in fat mass in both mice sexes, suggesting that TBT may affect fat deposition in a sex- and time-dependent manner in peripubertal and sexually mature mice (Penza et al., 2011).

White and brown adipose tissues (WAT and BAT) are complex endocrine organs that differ in morphology and their role in the metabolic function of mammals (Cinti, 2012). WAT is mainly composed of white adipocytes that are generally spherical in shape. Each contains a large, single lipid droplet that pushes all other organelles, including the nucleus, to the cell’s periphery (also a known as unilocular adipocyte), playing important role in energy storage and insulin sensitivity (Carobbio et al., 2017). BAT is composed of brown adipocytes that contain multiple lipid droplets dispersed throughout a more ellipsoidal-shaped cell that is enriched with iron-containing mitochondria, giving the cell and tissue a brownish hue (also a known as multilocular adipocyte). The thermogenic activity of brown adipocytes (heat production) is conferred by the presence of its numerous mitochondria containing uncoupling protein 1 (UCP-1) (Cypess et al., 2009). Recently, studies showed that “whitening” of BAT is an induction of white adipocyte features in brown adipocyte tissue, characterized by increased unilocular adipocyte depots, inflammatory cell infiltration, decreased expression of BAT metabolic markers, such as UCP1, PGC1α, beta-3 adrenergic receptor (Adrb3), etc, and a decreased number of intracellular lipid droplets and mitochondria (Wang et al., 2022; Guzzardi et al., 2022). Other studies also reported that “beiging” or browning of WAT by inducing brown adipocyte features in white adipocyte tissue could be inducible in both mice and humans by exposure to cold, diet, exercise, adipokines, chemical agents, or drugs (Ikeda et al., 2018; Kaisanlahti e Glumoff, 2019).

A few studies have reported that some obesogens can act, in part, by impairing the BAT thermogenic function, such as dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE) (La Merrill et al., 2014; vonderEmbse et al., 2021; Heindel e Blumberg, 2019). La Merril et al. (2014) showed that 1.7 mg/kg from gestational day 11.5 to postnatal day reduced body temperature, which became more pronounced with age, and impaired cold tolerance of female offspring at 6 months of age. This abnormal BAT function is caused, in part, by reduction in PGC1α and iodothyronine deiodinase 2 (Dio2), which converts T4 to the more thermogenic T3. Similarly, vonderEmbse et al. (2021) showed a reduction in body temperature in female mice at 4 months of age after perinatal exposure to 1.7mg DDT/kg and 1.3 mg p, p’-DDE/kg without BAT morphology abnormalities. Our results are consistent with these previous findings in that we observed a reduction in body temperature in room and cold condition TBT rats, which may be a consequence of changes in BAT morphophysiology. Cold exposure is responsible for induced BAT UCP1 expression to maintain normal temperature conditions in bodies of small rodents (Enerbäck et al., 1997; Ikeda et al., 2018). In our data, TBT exposure caused a borderline increase in BAT UPC1 protein expression after cold conditions.

To our knowledge, no studies have reported on the effects of subacute and low dose TBT exposure on BAT function for thermogenesis control. However, a few studies have reported some TBT consequences on BAT morphology and genes markers (Chamorro-García et al., 2013; Shoucri et al., 2018) (Chamorro-Garcia et al. 2013; Shoucri et al. 2018). Chamorro-Garcia et al. reported that exposure of pregnant F0 mice to three different concentrations of TBT (5.42, 54.2, or 542 nM) predisposed unexposed F3 male descendants to obesity and liver fat accumulation (Chamorro-García et al., 2013). Effects on depot size, adipocyte size, and the number of cells in BAT were modest in males. In particular, they observed increased lipid vesicles area, although not in all TBT exposure groups, presence of large lipid vesicles, and reduction in brown adipocyte numbers in F1, F2, and F3 males, although the effects were only significant in F2. Shoucri et al. (2018) found that production of beige/brite fat cells from mouse mesenchymal stem cells (MSC) was inhibited by TBT (50 nM) exposure for 2 weeks. Specifically, TBT induced lipid accumulation, reduced BAT markers such as UCP1 and PGC1α, and sustained the expression of fibrotic and inflammatory genes, repressing adipose browning process. Our results are consistent with these previous findings in that we observed an increase in BAT lipid accumulation and unilocular lipid adipocyte number, and reduction in multilocular adipocyte number in TBT rats at Room condition. However, TBT rats showed opposite responses in all these parameters in Cold condition, suggesting an intense remodeling of lipid droplets in the abnormal BAT adipocytes. Further, strong negative correlations were observed between BAT lipid accumulation and body temperature and multilocular adipocyte numbers. Thus, our data suggest that lipid accumulation changes the morphology of brown adipocytes that could be contributed to changes in their function to impair control of temperature in TBT rats.

BAT thermogenesis plays a critical role for the maintenance of body temperature in regular and cold circumstances in small rodents (Enerbäck et al., 1997). In cold conditions, brown adipocytes are activated by the sympathetic nervous system, inducing lipolysis. Fatty acids activate mitochondrial uncoupling protein 1 (UCP1) and are used as substrate for thermogenesis (Kajimura e Saito, 2014). In both humans and rodents, cold exposure induces proliferation of brown adipocyte and vascular endothelial cells, reflecting active angiogenesis that contributes to the enhancement of BAT function and whole-body thermogenic capacity (Klingenspor, 2003; Lee et al., 2015; Lim et al., 2012; Xue et al., 2009). Our results are consistent with these previous findings in that we observed an increase in the number of multilocular adipocyte in TBT rats in cold conditions, reflecting an attempt to maintain body temperature capacity. However, we did not find an increase in blood vessels number in BAT of TBT rats in cold conditions, only in CON rats, suggesting a possible impairment blood flow in BAT of TBT rats.

Inflammation is a common feature in abnormal adipose tissue as result of TBT exposure or obesity models (Freitas-Lima et al., 2018; Mirzaei et al., 2013; Ravanan et al., 2011). Previously, we reported that mast cells are crucial inflammatory cells present in abnormal fat after TBT exposure and linked with metabolic complications in female rats (Bertuloso et al., 2015; Freitas-Lima et al., 2018; da Costa et al., 2019). Similarly, Liu et al. (2009) reported that genetic deficiency of mast cells can lead to increased energy expenditure and promote UCP-1 protein expression in BAT from male obese mice induced by Western diet for 12 weeks. We observed an increase in mast cell number in BAT of TBT rats in Room condition. In addition, a strong positive correlation was observed between BAT lipid accumulation and mast cell number at room temperature, suggesting that the presence of mast cells could play some role in abnormal BAT with more lipid accumulation. However, it is important to note that mast cells are cold sensors, becoming degranulated when exposed to cold. This results in histamine release that increases blood flow in BAT, which is an important physiologic response for maintaining proper thermogenesis (Desautels et al., 1994; Finlin et al., 2019; Rothwell et al., 1984; Shen et al., 2008). We observed an increase in mast cell number that was linked with high blood vessel number in BAT of control rats in cold conditions, suggesting that a possible increase in blood flow in BAT of control rats. In addition, a strong positive correlation was observed between BAT lipid accumulation and mast cell number in cold conditions. It has been frequently noted that the infiltration of macrophages or other inflammatory cells accompany the ‘whitening’ of brown fat, suggesting a role of inflammation in mediating impairments in BAT function in male mice (Kotzbeck et al., 2018). We observed an increase in ED1 protein expression (macrophage marker) in BAT of TBT rats after cold condition. Moreover, a negative correlation was observed between BAT multilocular adipocyte number and mast cell number in room temperature conditions, suggesting that an increase in presence of mast cells is associated with a reduction in BAT multilocular adipocyte number. Thus, these inflammatory makers could contribute to changes in BAT function leading to abnormal control of temperature in TBT rats.

Other adipose tissue abnormalities were observed in TBT models, including fat oxidative stress and fibrosis (Freitas-Lima et al., 2018; da Costa et al., 2019). Our current results are consistent with these previous findings as we observed an increase in BAT collagen in both room temperature and cold conditions. TBT exposure also caused a borderline increase in BAT COL1A1 protein expression in cold condition compared to respective control. We also observed a negative correlation between body temperature and COL1A1 protein expression at room condition, suggesting these BAT changes increase collagen deposition and contribute to reduction in thermogenesis function to control body temperature. Oxidative stress could be a consequence of inflammatory cell actions, metabolization of TBTs, changes in antioxidant genes, abnormal morphology conditions, etc (da Costa et al., 2019). Our current results are consistent with these previous findings as we observed an increase in BAT oxidative stress in both room temperature and cold conditions. Strong positive correlations were observed between BAT oxidative stress and mast cell number at room temperature, suggesting that the presence of mast cells could play some role in oxidative stress in BAT of TBT rats. Further, BAT oxidative stress was positively correlated with lipid accumulation, suggesting increase in lipid peroxidation of lipid storage, leading to impairment in BAT function to control temperature in TBT rats. These data suggest that TBT exposure can lead to BAT irregularities, which can be replaced by fibrous tissue, leading to increases in collagen deposition.

Our study provides evidence that subacute low dose TBT exposure for 15 days is responsible for reduction in maintenance of body temperature in room and cold condition in male rats. TBT exposure alters BAT morphology involved lipid accumulation, lipid droplet remodeling, inflammation, fibrosis, and oxidative stress (Table 1). Collectively, these data indicate that TBT exposure alters BAT morphology and gene expression that contribute to abnormal thermogenesis control in male rats. Exposure to TBT does not present a pattern of effects in a dose dependent manner, showing a non-linear dose response, similar the actions of other EDCs that lead to U-shaped or inverted U-shaped dose response curves (Gore et al., 2015). Thus, in future studies, it is necessary to analyze the effects of TBT for prolonged periods on female BAT. Furthermore, since abnormal BAT morphology could interfere with metabolic functions, it is important for future studies to investigate whether the mixture affects female metabolic parameters in vivo.

Supplementary Material

1

Fig. S1. BAT collagen deposition evaluation in male rats. Representative Masson’s trichrome staining in BAT of CON (A) and TBT (B) rats from room temperature condition, and CON (C) and TBT (D) rats from cold condition. (E) BAT collagen deposition evaluation, n=5–6.

2

Fig. S2. Body weight, adiposity, EAT weight and morphology, and adipokines levels in male rats. (A) Body weight, (B) EAT weight and (C) adiposity in CON and TBT rats. Representative H&E-stained in EAT of CON (D) and TBT rats (E). (F) EAT adipocytes diameter. Representative Alcian Blue-stained in EAT CON (G) and TBT (H). (I) EAT mast cells number. (J) EAT MPO activity. (K) EAT NAG activity. Serum (L) adiponectin and (M) leptin levels. Student’s t-test. Arrowhead: Mast cell; MPO: Myeloperoxidase (neutrophil presence); NAG: N-Acetyl-β-D-Glucosaminidase (macrophage presence); EAT: epidydimal adipose tissue. n=5–10

3

Fig. S3. Glucose metabolism and pancreas morphology evaluation in male rats. (A) GTT, (B) AUC of GTT, (C) IST, (D) AUC of IST, (E) serum insulin levels in CON and TBT rats. Representative H&E-stained of pancreas in CON (F) and TBT rats (G). (H) Pancreas islet number. Representative Alcian Blue-stained in pancreas CON (I) and TBT rats (J). (K) Pancreas mast cells number. (L) Pancreas MPO activity. (M) Pancreas NAG activity. Student’s t-test. Arrowhead: Mast cell; GTT: Glucose tolerance test; IST: Insulin sensibility test; MPO: Myeloperoxidase; NAG: N-Acetyl-β-D-Glucosaminidase. n=5–6

4

Fig. S4. Liver function and morphology evaluation in male rats. (A) Serum GPT activity and (B) serum GOT activity in CON and TBT rats. Representative H&E-stained in liver of CON (C) and TBT rats (D). Representative Alcian Blue-stained in liver of CON (E) and TBT rats (F). (G) Liver mast cells number. (J) Liver MPO activity. (K) Liver NAG activity. ****p < 0.0001 vs CON. Student’s t-test. Arrowhead: Mast cell; GOT: glutamic oxaloacetic transaminase; GPT: Glutamate Pyruvate Transaminase; MPO: Myeloperoxidase; NAG: N-Acetyl-β-D-Glucosaminidase. n=5–6.

5

Fig. S5. Thyroid function and morphology evaluation in male rats. Representative H&E-stained in thyroid of CON (A) and TBT rats (B). (C) Thyroid follicles diameter and (D) thyroid follicles number. Representative Alcian Blue-stained in liver of CON (E) and TBT rats (F). (G) Thyroid mast cells number. (H) Serum free T4 levels. (I) Serum total T3 levels. *p < 0.05 vs CON. Student’s t-test. Arrowhead: Mast cell; T3: Triiodothyronine; T4: Thyroxine. n=5–6.

Highlights.

• TBT exposure impairs thermogenic function

• TBT exposure led to BAT lipid accumulation

• TBT exposure led to BAT inflammation

• TBT exposure led to BAT oxidative stress

• TBT exposure led to BAT fibrosis