ELSEVIER
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
-
Science OF THE Total Environment
Insight into the endocrine disrupting effect and cell response to butyltin compounds in H295R cell: Evaluated with proteomics and bioinformatics analysis
Check for updates
Xueting Yan a,b, Bin He a,b,*, Ligang Hu a,c,*, Jiejun Gao a,b, Shuai Chen ª, Guibin Jiang a,b
a State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b University of Chinese Academy of Sciences, Beijing 100049, China
” Institute of Environment and Health, Jianghan University, Wuhan, Hubei 430056, China
d Department of Radiation Oncology, Washington University in St. Louis, 4511 Forest Park Ave, St. Louis, MO 63108, USA
HIGHLIGHTS
· The endocrine disrupting effect of BTs in H295R was investigated at environmen- tally relevant concentration.
· Exposure of BTs induced perturbation in expression of proteins related to hor- mone and lipid metabolism.
· BTs can activate nuclear receptor path- ways and modulate cellular metabolism homeostasis.
· TBT was more potent than DBT and MBT in H295R.
ARTICLE INFO
Article history: Received 11 December 2017 Received in revised form 10 February 2018 Accepted 13 February 2018 Available online 20 February 2018
Editor: Henner Hollert
Keywords: Butyltin compounds H295R cell Endocrine disruption
Proteomics Bioinformatics analysis
GRAPHICAL ABSTRACT
H295R cell
2-DE-MALDI-TOF-MS
TBT DBT MBT
Nuclear receptor activation/lipid metabolism disruption
LXRIRXR Activation
Renal Necrosis/Cell Death
> LXR/RXR activation
FXBUIRXR Activation
> FXR/RXR activation
> Fatty acid metabolism
Cardiac Necrosis/Cell Death
4og10[P Value)
Fatty Acid Metabolism
Bioinformatics analysis
ABSTRACT
The widespread use of organotin compounds (OTs) as biocides in antifouling paints and agricultural applications poses a serious threat to the ecosystem and humans. Butyltin compounds (BTs), especially tributyltin (TBT), are con- sidered to be endocrine disrupting chemicals in marine organisms. The underlying mechanism of disrupting effects on mammals, however, has not been sufficiently investigated. To determine the effect and action of these biocides, the present study evaluated the effects of BTs on human adrenocortical carcinoma cells (H295R) with a focus on en- docrine disrupting effect. Two-dimensional electrophoresis (2-DE) and subsequent mass finger printing were used to identify proteins expression profiles from the cells after exposure to 0.1 µM BTs for 48 h. In total, 89 protein spots showed altered expression in at least two treatment groups and 69 of these proteins were subsequently identified. Bioinformatic analysis of the proteins indicated that BTs involved in the regulation of hormone homeostasis, lipid me- tabolism, cell death, and energy production. IPA analysis revealed LXR/RXR (liver X receptor/retinoid X receptor) ac- tivation, FXR/RXR (farnesoid X receptor/retinoid X receptor) activation and fatty acid metabolism were the top three categories on which BTs acted and these systems play vital roles in sterol, glucose and lipid metabolism. The expres- sion of LXR and FXR mRNA in H295R cells was stimulated by TBT, confirming the ability of TBT to activate this nuclear receptor. In summary, the differentially expressed proteins discovered in this study may participate in the toxic ac- tions of BTs, and nuclear receptor activation and lipid metabolism may play important roles in such actions of BTs. @ 2018 Elsevier B.V. All rights reserved.
* Corresponding authors at: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, No 18 Shuangqing Road, Haidian, Beijing 100085, China. E-mail addresses: bhe@rcees.ac.cn (B. He), lghu@rcees.ac.cn (L. Hu).
1. Introduction
Organotin compounds (OTs), mainly butyltin compounds (BTs), are widely used as antifouling paints, agricultural pesticides, catalysts, sta- bilizers and preservatives in multiple industries since their initial syn- thesis in 1849 (Antizar-Ladislao, 2008). Although they were banned in maritime applications in 2008, contamination with BTs is still wide- spread due to their persistence in the environment and growing de- mand for their use in industry and agriculture. Concentrations of BTs up to 166.5 ng (Sn) g-1 in surface sediment and from 29.4 ng (Sn) g-1 to 206.0 ng (Sn) g-1 in bivalve mollusk were found in the Pata- gonian coastal zone (Commendatore et al., 2015). A study on seafood contamination by OTs in Hong Kong reported that tributyltin (TBT) occurred in the tissues of Collichthys lucidus and Harpadon nehereus at concentrations of 62.9 and 105.3 ug kg-1 dry weight (dw), respec- tively (Ho and Leung, 2014).The use and release of OTs pose serious threats to the ecosystem and humans through the food chain transfer (Jiang et al., 2001; Cao et al., 2009).
Several studies have reported that BTs can induce genotoxic, neuro- toxic, hepatotoxic, teratogenic, and immunosuppressive toxicities, as well as imposex in various organisms (Cima and Ballarin, 2012; Graceli et al., 2013; Pagliarani et al., 2013; Zhou et al., 2002). Among the BTs, TBT has been demonstrated to affect basic procreation functions and interfere with hormone secretion, and is considered an endocrine disrupter (Omura et al., 2001). Accumulation of OTs in seafood can dis- rupt reproductive functions in female rats (Podratz et al., 2015). The mechanism of endocrine disruption caused by BTs (such as TBT) is in- complete; however, interference with steroidogenic pathways and re- lated enzymes including cytochrome P450 isomers (CYP) appears to be associated with endocrine effects (Sanderson et al., 2002; Nakanishi, 2008; Oberdorster and McClellan-Green, 2002; Cao et al., 2017). TBT has also been reported to be a potent inducer of adipogenesis (Chamorro-García et al., 2013). Bertuloso et al. suggest that TBT leads to adiposity in white adipose tissue, which is associated with inflamma- tion and estrogen receptor alpha (ERa) pathways (Bertuloso et al., 2015). Another study reported that TBT contributed to disruption of proper functioning of the hypothalamic-pituitary-gonadal axis, leading to obesity and abnormal kisspeptin/leptin signaling in female rats (Sena et al., 2017). Dibutyltin (DBT) is commonly considered to have the greatest immunotoxicity of the OTs, as it binds to glucocorticoid re- ceptors. TBT and DBT have been reported to alter the secretion of tumor necrosis factor (TNF) « and interferon gamma (IFNy) in human im- mune cells (Lawrence et al., 2015). Monobutyltin (MBT) has relatively lower toxicity and is generally detected along with TBT and DBT in the environment. BTs have also been shown to induce apoptosis, which generally occurs via induction of an intracellular rise in Ca2+, oxidative damage and endoplasmic reticulum (ER) stress (Mitra et al., 2013). The toxicity of BTs has been confirmed in various cell lines, mammals and marine organisms, but the underlying molecular mechanisms of their effects and action have not been clearly elucidated (Leung et al., 2006; Mitra et al., 2015; Ferreira et al., 2013; Zhang et al., 2013). To illustrate the action mechanism of BTs, TBT, DBT and MBT were screened to ex- amine the effects on human cells, and then explore the relationship be- tween the protein profiles and related pathways.
Human adrenocortical carcinoma cells (H295R) were used as the cell model and are angiotensin-II-responsive steroid producing adreno- cortical cells that maintain expression of genes, enzymes and steroido- genic hormones (Gazdar et al., 1990). In addition, H295R cells have a good correlation with the toxic responses seen in normal adult human adrenal cells. The H295R steroidogenesis assay has been developed and validated in comprehensive studies by the Organization for Eco- nomic Co-operation and Development (OECD) guideline (Hollert and Giesy, 2007; Hecker et al., 2011). It’s a good cell line for evaluating the chemical effects on gene expression, enzymatic activity and hormone production, as well as on proteome changes (Hecker et al., 2006; Sanderson, 2006).
Proteomic approaches can provide information on the global re- sponses of model systems towards the toxins. Together with bioinfor- matic approaches, comprehensive insights into the mechanisms of toxic effects can be obtained. These approaches have been used to inves- tigate the toxic effects of substances such as perfluorooctane sulfonate, mycotoxin zearalenone, «- and ß-zearalenol, and carbonyl sulfide (Shi et al., 2009; Busk et al., 2011; Busk et al., 2012; Lardinois et al., 2014). Using these approaches a two-dimensional gel electrophoresis-matrix assisted laser desorption ionization mass spectrometry (2-DE-MALDI MS)-based proteomic analysis was performed to reveal the nanoparticle-specific effects of silver nanoparticles on Caco-2 cells com- pared with ionic silver (Oberemm et al., 2016). Monitoring protein ex- pression changes under exposure to chemical contaminants is an effective way to characterize the important molecular targets and help to understand the mechanism of actions.
In this study, we aimed to explore the toxic effect and molecular mechanism of various BTs on H295R cells, with a focus on endocrine disrupting effect. A proteomic response to BT exposure was investigated with a comparative proteomic approach. Further bioinformatic analysis was performed to explore the activation of nuclear receptor and meta- bolic pathways in H295R cells caused by exposure to BTs. Our data showed that BTs activated LXR/RXR (liver X receptor/retinoid X recep- tor) and FXR/RXR (farnesoid X receptor/retinoid X receptor) and thereby modulate cellular sterol and lipid homeostasis.
2. Materials and methods
2.1. Chemicals
TBT, DBT and MBT, were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Stock solutions for BTs were prepared by dissolv- ing the compounds in ethanol to a final concentration of 1.0 mg mL-1. All chemicals used for electrophoresis were purchased from GE Healthcare Bio-Sciences (Uppsala, Sweden). Ultrapure water (18.2 MQ2 cm) was prepared using a Milli-Q Advanced A10 system (Millipore, Bedford, USA).
2.2. Cell culture
The human adrenocortical carcinoma cell line H295R (ATCC, CRL- 2128, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F-12, HyClone) supplemented with 1% insulin-transferrin-selenium-G (ITES-G, Gibco), 1% penicillin- streptomycin (Gibco), and 2.5% Nu-Serum (BD Bioscience) at 37 ℃ with 5% CO2. The medium was changed three times per week and cells were trypsinized once a week for subculturing. Cells were used be- tween passages 5 and 15.
2.3. Cell viability assay and exposure
BTs were diluted with absolute ethanol and added to cell cultures at a final ethanol concentration of 0.1% (v/v). Solvent controls contained the same amount of ethanol as the BT dilutions. H295R cells were seeded at a density of 3 x 105 cells mL-1 in a 96-well plate and incu- bated for 24 h before exposure to BTs. After 24 h of pre-incubation, cells were exposed to 0.01, 0.05, 0.1, 0.5, 1 and 5 uM of the three tested BTs (TBT, DBT and MBT) and the medium was exchanged with phenol red-free DMEM/F-12 medium (HyClone) supplemented with 1% ITES- G, 1% penicillin-streptomycin, and 2.5% charcoal stripped fetal bovine serum (Biological industries). After 48 h of exposure, 100 uL of medium was discarded and 10 uL resazurin was added to per well to a final con- centration of 10 uM. Cells were then incubated under the same condi- tions for 2 h, and the fluorescence signal (530 nm/590 nm, excitation/ emission) was measured using a spectral scanning multimode plate reader (Thermo Scientific Varioskan Flash). Viability of the H295R
cells was evaluated using the Alamar Blue Assay (Invitrogen, Carlsbad, CA) and expressed as percentage of the solvent control.
2.4. Protein extraction
Protein extraction was performed by cell lysis after exposure to 0.1 uM (sublethal concentration) BTs for 48 h. The BT concentrations used here were comparable with the concentrations (65 nM) found in human blood samples from Michigan, USA (Kannan et al., 1999). Ten million cells were added to 500 µL lysis buffer, which contained 7 M urea, 2 M thiourea, 4% w/v 3-[(3-chola-midopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiothreitol (DTT), 1% protease inhibitor cocktail, 0.5 uL benzonase, and 2% IPG buffer (3-10, Non-linear). The entire sample was then collected and transferred to a 1.5 mL centrifuge tube using a cell scraper. Samples were incubated on ice for 1 h and then centrifuged at 12,000g for 10 min at 4 ℃. The supernatants were collected in new 1.5 mL centri- fuge tubes and 10 uL was to determine the concentration using a 2-D Quant Kit (GE Healthcare). Samples were stored at -80 ℃ for further use.
2.5. Separation of proteins with 2-DE
2-DE analysis was based on the procedure described by Shi et al., 2009. Protein samples (approximately 350 µg protein on each gel) were diluted with rehydration buffer (8 M urea, 2% w/v CHAPS, 65 mM DTT, 0.5% IPG buffer, 0.002% v/v bromophenol blue) to a final volume of 350 µL. Rehydration and isoelectric focusing (IEF) were car- ried out automatically on pre-prepared immobilized gradient (IPG) strips (pH 3-10, Non-linear, 24 cm, GE Healthcare) at 30 V for 6 h, 60 V for 6 h, 500 V for 1 h, 1000 V for 1 h, 8000 V for 1 h and then 8000 V until to a total of 64 kVh on a Multiphor II system (GE Healthcare). The entire procedure was performed at 20 ℃ (Görg et al., 1991). After focusing, the strips were firstly equilibrated in IPG equili- bration buffer (50 mM Tris-HCl pH 8.8, 30% v/v glycerol, 6 M Urea, 2% w/v sodium dodecyl sulfate (SDS) and 0.002% v/v bromophenol blue) with 20 mg mL-1 DTT for 15 min and then in IPG buffer with 25 mg mL-1 iodoacetamide instead of DTT for a further 15 min (Görg et al., 2004). After equilibration, the IPG strip was placed on top of a 12.5% homogeneous polyacrylamide gel and fixed in 0.5% agarose for second dimension separation with an Ettan DALT II system (GE Healthcare). Tris glycine SDS buffer (25 mM Tris, 198 mM glycine, and 0.1% SDS) was used for the second dimensional separation. Electropho- resis was performed at 5 W/gel for 40 min and then at 10 W/gel until the bromophenol blue reached the bottom of the gel. To obtain better reso- lution, the running temperature of the electrophoresis was maintained at 20 ℃ with a thermostat (Görg et al., 1991). Protein spots on the gels were visualized with silver staining after fixing in ethanol/acetic acid/water (4:1:5 v/v/v) overnight (Görg et al., 1988).
2.6. Image acquisition and protein identification
Gel images were obtained with an Image Scanner (GE Healthcare). Image Master™ 2D Platinum software (Amersham Biosciences) was used to detect and quantify protein spots on the gel images. The area and intensity of each protein spot was expressed as the volume of the spot, which was compared with the total volume of the matched spots in the gel. Only spots with >1.5-fold increase or <0.67-fold decrease in size between the BT-treated and control groups were considered as up- or down-regulated spots (Liu et al., 2015).
Protein spots of interest were excised from the gel and washed twice using ultrapure water for 5 min at 37 ℃. Spots were then destained, acetonitrile-dehydrated and spun dry for 5 min under vacuum. After drying, each gel piece was rehydrated in 25 mM ammonium bicarbon- ate containing 10 ng µL-1 trypsin and then incubated overnight at 37 °℃ to digest proteins (Pandey and Mann, 2000). The identification
of proteins were performed on an Autoflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany) equipped with a 200-Hz smartbeam laser (Gamberi et al., 2015). Protein identifica- tion was carried out using the peptide mass fingerprint (PMF) as an input to search the human (Homo sapiens) sub-database of the Uniprot protein database.
2.7. Bioinformatic analysis
Information on protein classification and function were analyzed by Gene Ontology (GO) (www.geneontology.org). Pathways were eluci- dated according to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway (http://www.genome.ad.jpkegg/pathway.html) associated with each differentially expressed protein. Ingenuity Pathway Analysis (IPA, Ingenuity Systems, www.ingenuity.com) software was used to construct a functional network based on the gene names of the affected proteins. The top categories affected by BTs were also determined by IPA.
2.8. Western blotting analysis
In order to confirm the protein expression determined by 2-DE, 17- beta-hydroxysteroid dehydrogenase 10 (HSD17B10), fatty acid-binding protein, epidermal (FABP5) and 14-3-3 protein sigma (SFN) were se- lected for expression analysis by western blotting. H295R cells in 6- well plates were exposed to 0.1 µM MBT, DBT and TBT for 48 h and ve- hicle treated cells were used as a control. After incubation, the cells were washed three times with cold phosphate buffered solution (PBS) and extracted using RIPA lysis buffer (Sigma-Aldrich, USA) with protease in- hibitors (EDTA-free Protease Inhibitor Cocktail Tablet, Roche, USA). Pro- tein concentration was quantified by Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific, USA). Equal amounts of whole pro- tein extracts from different groups were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incu- bated with blocking buffer (Tris Buffered Saline with Tween 20 (TBST) with 5% milk) for 1 h and then blocked overnight at 4 ℃ with the pri- mary antibodies HSD17B10 (1:300, AbCam, UK), FABP5 (1:1000, AbCam, UK) or SFN (1:1000, Bio-Rad, USA). After washing three times in TBST, the membranes were then incubated for 1 h with a horseradish peroxidase-conjugated secondary anti-IgG antibody (1:1000, AbCam, UK) in TBST. Protein bands were detected using the chemiluminescence (ECL) system (Thermo Scientific, USA). Band density was determined with Quantity One software (Bio-Rad).
2.9. RNA isolation and real-time PCR (qPCR)
H295R cells were treated with BTs for 48 h. Total RNA was then iso- lated using TRIzol RNA Isolation kit (Invitrogen) following the manufac- turer’s instructions. Reverse transcription was performed using the PrimeScript RT Master Mix (Takara, Japan) according to the manufac- turer’s protocol. Real-time quantitative PCR reactions (95 ℃, 3 min for 1 cycle; 95 ℃, 15 s; 60 ℃, 30 s; 72 ℃, 30 s for 40 cycles) were performed using SYBR Green PCR Mix (Applied Biosystems) and the real-time PCR detection system (Bio-Rad, CFX-96, USA). The primer sequences for LXRa, LXRØ, FXR and B-actin are listed in Table S2. Relative levels of gene expression in comparison with ß-actin (housekeeping gene) were determined using the 44Ct method (Livak and Schmittgen, 2001). Three individual experiments were performed.
2.10. Statistical analysis
Student’s t-test was used to determine significant differences in pro- tein expression between control and treatment groups. P < 0.05 was considered to be statistically significant.
3. Results
3.1. Cell viability
Cell viability was investigated after exposing H295R cells to a series of concentrations of BTs. All treated groups showed concentration- dependent responses in viability, where cells exposed to 0.1 µM main- tained >90% viability compared to controls (Fig. S1). At 0.5 uM, the treated groups showed significantly lower viability, especially in the TBT group. Cellular viability after exposure to 0.5, 1 and 5 uM TBT de- creased to 60%, 30% and 13%, respectively. The viability of the H295R cells was reduced to 57% and 35% by 1 and 5 uM DBT, while high doses of MBT (0.5, 1 and 5 uM) tended to give relatively moderate de- creases in cell viability to 77%, 78% and 73%, respectively. The lowest ef- fect concentrations (LEC) of TBT, DBT and MBT were 0.5 uM, 1 µM and 5 uM, respectively, indicating that TBT was the most toxic agent followed by DBT and MBT. Furthermore, morphologic changes were ob- served in TBT treated cells, which showed that at high concentrations cell crenation and adhesion rates decreased significantly (Fig. S2).
3.2. Effects of BTs (TBT, DBT and MBT) on the proteome of H295R cells
Based on the cell viability results, a concentration of 0.1 µM of BTs was chosen for proteome investigations. Total proteins extracted from different groups were stained with silver. 2-DE gels for BTs and controls are shown in Fig. 1 (A-D). >1800 protein spots were separated on each
gel. Remarkably, all BT treatments caused moderate alterations in pro- tein expression profiles. When a threshold of >1.5-fold increase or <0.67-fold decrease in expression was used, 89 protein spots were found to be affected by BTs (in at least two treatment groups) compared with the controls. Among the 89 protein spots, 69 were tentatively iden- tified, which included 32 up-regulated and 37 down-regulated proteins. Not all differentially expressed spots could be identified due to low con- centrations of proteins, manual extraction errors and MS sensitivity lim- itations. The identified proteins are listed in Table S1. The positions of the 69 identified proteins are illustrated by arrows and numbers in the representative gel (Fig. S3).
Fold-change values for the three BT treatment groups after normal- ization against the solvent control are shown in Fig. 2. It was apparent that the effects on protein expression changes were most pronounced in the TBT treated group followed by cells treated with DBT. The MBT treated groups showed the smallest number of changes compared with the other two treated groups.
3.3. Classification of differentially expressed proteins
To understand the biological functional changes associated with pro- tein expression after exposure to BTs, all identified proteins were clus- tered into functional categories based on GO terms. The proteins were classified based on three major criteria: molecular functions, biological processes and cellular components shown in Fig. S4. According to the molecular functions criteria, most of the regulated proteins were
A. Control
B. TBT
C. DBT
D. MBT
A. TBT
categorized as binding (66.7%), catalytic activity (37.7%), enzyme regu- lator activity (16.0%) and receptor activity (10.0%). The remaining iden- tified proteins were shared among the categories of transporter activity, electron carrier activity, antioxidant activity, signal transducer activity, and structural molecule activity, which make up 18.8% of the proteins in total (Fig. S4A). The regulatory proteins were involved in biological processes including single-organism processes (63.8%), response to stress (39.1%), small molecule metabolic processes (37.7%), transport (33.3%), localization (29.0%), regulation of protein metabolic processes (26.1%), negative regulation of metabolic processes (26.1%), oxidation- reduction processes (23.2%) as well as cell death (23.2%) (Fig. S4B). From the aspect of cell components, the identified proteins belonged to cell (63.8%), organelle (53.6%), cytoplasm (37.7%), extracellular re- gion (33.3%), membrane-enclosed lumen (29.0%), membrane (26.1%) and macromolecular complex (20.3%) (Fig. S4C).
3.4. KEGG pathways analysis
Generally, proteins were mapped to KEGG pathways based on their corresponding gene names. The results (Fig. 3B) indicated that twenty- three pathways associated with metabolism, genetic information pro- cessing, environmental information processing, organismal systems and human diseases were significantly affected by BTs. From these path- ways the representative metabolic pathways included glycolysis/gluco- neogenesis, carbon metabolism, amino acid metabolism, fatty acid metabolism, as well as steroid hormone biosynthesis.
3.5. Ingenuity pathway analysis for top toxic lists and gene interaction network In the list of most affected categories (according to the P-value), LXR/ RXR activation, FXR/RXR activation, fatty acid metabolism, renal
| 0.125 | Fold Change 0.25 0.5 8 4 2 1 | 0.125 Fold Change 0.25 0.5 4 2 1 | 8 0.125 | Fold Change 0.25 0.5 8 2 4 1 | ||||
|---|---|---|---|---|---|---|---|---|
| 191 209 | C. 191 209 | B. 191 209 | ||||||
| 236 | 236 | |||||||
| 255 256 270 287 | 255 | 236 | ||||||
| MBT 256 270 | DBT 255 256 270 | |||||||
| 310 403 | 287 310 | 287 310 | ||||||
| 467 468 | 403 467 468 | 403 467 | ||||||
| 469 470 | 469 | 468 469 | ||||||
| 476 | 470 476 | 470 | ||||||
| 478 | 478 | 476 | ||||||
| 479 | 479 | 478 | ||||||
| 481 | 481 | 479 | ||||||
| 497 | 497 | 481 497 516 527 564 578 622 645 650 | ||||||
| 516 527 564 578 622 645 650 657 662 | 516 527 564 578 622 645 650 657 | |||||||
| 666 671 | 662 666 | 657 662 | ||||||
| 691 694 724 | 671 691 694 | 666 671 691 | ||||||
| 731 742 750 759 780 | 724 731 742 750 759 | 694 724 731 742 750 | ||||||
| 784 789 803 815 | 780 784 789 | 759 780 784 789 | ||||||
| 824 829 834 860 | 803 815 824 829 834 | 803 815 824 829 | ||||||
| 903 939 | 860 903 | 834 860 | ||||||
| 973 978 | 939 973 | 903 939 973 | ||||||
| 1002 | 978 | 978 | ||||||
| 1042 | 1002 | 1002 | ||||||
| 1077 1088 1132 | 1042 1077 1088 | 1042 1077 1088 | ||||||
| 1146 | 1132 1146 | 1132 | ||||||
| 1174 | 1174 | 1146 | ||||||
| 1297 | 1297 | 1174 | ||||||
| 1319 1431 | 1319 | 1297 | ||||||
| 1463 | 1431 | 1319 1431 | ||||||
| 1472 | 1463 | |||||||
| 1486 | 1472 | 1463 | ||||||
| 1493 | 1486 | 1472 | ||||||
| 1551 | 1493 | 1486 | ||||||
| 1554 | 1551 | 1493 | ||||||
| 1570 | 1554 | 1551 | ||||||
| 1612 | 1570 | 1554 | ||||||
| 1614 | 1612 | 1570 1612 | ||||||
| 1620 | 1614 | 1614 | ||||||
| 1620 | 1620 | |||||||
Spot Number
necrosis/cell death and cardiac necrosis/cell death were the top five rel- evant targets for cell injury induced by BTs (Fig. 3A). IPA identified 5 gene networks with scores ranging from 14 to 41, of which the top 3 were associated with: 1) Developmental disorders, hereditary disor- ders, metabolic diseases (score = 41) (Fig. S5A); 2) Cell death and sur- vival, organismal injury and abnormalities, metabolic diseases (score = 33) (Fig. S5B); and 3) Hematological system development and function, immunological diseases, lymphoid tissue structure and development (score = 23) (Fig. S5C).
3.6. Western blotting
The antibodies used for Western blotting were selected on the basis of the proteins role in hormone homeostasis (HSD17B10), lipid metab- olism (FABP5), and apoptosis (SFN). The level of protein alterations ob- served by Western blotting showed a similar trend compared with the changes detected by 2-DE (Fig. 4). Thus, Western blotting analysis of HSD17B10, FABP5 and SFN on the fraction of H295R cells exposed to BTs confirmed the changes detected by 2-DE and mass spectrometry. The expression of GAPDH was independent in each group and was used as a normalization control.
3.7. Expression of LXR and FXR in H295R cells treated with BTs
To further investigate the mechanisms of the effect of BTs and their modulation of LXR and FXR mRNA, RT-PCR was performed. As shown in Fig. 5, TBT significantly increased the expression of LXRa and FXR mRNA, while the LXRØ mRNA expression did not change significantly. The results show that DBT and MBT have no effect on LXR and FXR in
H295R cells.
A. Top toxic Analysis
B. KEGG Pathways Analysis
Glycolysis / Gluconeogenesis
Carbon metabolism
Valine, leucine and isoleucine degradation
Fatty acid metabolism
LXR/RXR Activation
Metabolic pathways
Complement and coagulation cascades
Proteasome
Fructose and mannose metabolism
Renal Necrosis/ Cell Death
Pentose and glucuronate interconversions
FXR/RXR Activation
Steroid hormone biosynthesis
Fatty acid degradation
Nitrogen metabolism
1
Metabolism of xenobiotics by cytochrome P450
Fatty acid elongation
Antigen processing and presentation
beta-Alanine metabolism
Cardiac Necrosis/ Cell Death
9 -log10 (P Value)
Fatty Acid Metabolism
Tyrosine metabolism
Prion diseases
Riboflavin metabolism
D-Glutamine and D-glutamate metabolism
Pyruvate metabolism
HIF-1 signaling pathway
Drug metabolism - cytochrome P450
0
5
10
15
-log10 (P Value)
Control MBT DBT TBT
HSD17B10
FABP5
SFN
GAPDH
3.5
*
T
HSD17B10
3.0
FABP5
SFN
*
2.5
Fold Change
*
*
2.0
T
1.5
T
1.0
T
T
T
I
T
0.5
* T
0.0
Control
MBT
DBT
TBT
Control MBT DBT TBT
Control
MBT
DBT
TBT
4. Discussion
In this study, we evaluated the toxicity of BTs, as endocrine disrupting chemicals, to modulate protein expression profiles and acti- vate nuclear receptors in H295R cells. Although peroxisome proliferator activating receptor y (PPARy) and RXR have been demonstrated to in- teract with TBT (Nakanishi, 2008), we found that two other nuclear re- ceptors (LXR and FXR) were also activated by TBT. In the present study, we showed that TBT at up to 0.1 uM significantly increased the
3.0
Control
MBT
2.5
DBT
mRNA relative expression
TBT
*
2.0
*
T
1.5
T
Y
T
1.0
T
T
T
T
T
T
0.5
0.0
LXRa
LXR₿
FXR
expression of LXRa and FXR mRNA, indicating that TBT acts as a poten- tial agonist for LXR&/RXR and FXR/RXR.
A substantial amount of proteins appeared to be sensitive to the BTs treatment in H295R cells. The identified 69 proteins were mainly re- lated to cellular homeostasis, energy metabolism, stress response, apo- ptosis, transport and a number of human disease pathways. IPA analysis showed that LXR/RXR activation, FXR/RXR activation, fatty acid metabolism and cellular necrosis were the main affected pathways or action targets of BTs in H295R cells (Fig. 3). Nuclear receptors partic- ipate in the translation of hormonal, metabolic and nutritional signals, helping to maintain bodily homeostasis (Calkin and Tontonoz, 2012). Two isotypes of the LXRs exist, namely LXRa and LXRØ, and these have been found to be important in the regulation of cholesterol, fatty acid, and glucose homeostasis (Tang et al., 2014). FXR is a nuclear bile acid receptor that plays a pivotal role in bile acid homeostasis, lipid and glucose metabolism (Cai et al., 2010). Both LXR and FXR can form obligate heterodimers with RXR to modulate the expression of target genes, and LXR/RXR and FXR/RXR can also be activated by RXR ligands (Calkin and Tontonoz, 2012). TBT and triphenyltin (TPT) have been re- ported to be high affinity ligands for RXR and binding of these com- pounds caused the development of imposex in female rock shells (Nishikawa et al., 2004). Further studies have demonstrated that TBT acts as an obesogen through the nuclear receptors PPARy and RXR to promote adipogenesis and alter lipid homeostasis in vitro and in vivo (Grün et al., 2006; Kanayama et al., 2005). Similarly to TBT, DBT has also been found to be a partial RXRox agonist with significant adipogenic potential (Milton et al., 2017). In our study, LXR/RXR activation and FXR/RXR activation were two targets affected by BTs in H295R cells. To address if BTs can activate LXR and FXR, we evaluated the mRNA ex- pression levels of LXRo, LXRØ and FXR. The qPCR results showed that the expression of LXRa and FXR mRNA in H295R cells was induced by TBT (Fig. 5). Moreover, there were no significant changes in LXRa, LXRØ and FXR mRNA due to treatment with DBT or MBT. Thus, TBT can activate LXR/RXR and FXR/RXR by interacting with LXRox and FXR.
BTs have been reported to induce imposex in marine mollusks and act as endocrine disrupters in a variety of taxa, including amphibians, fish, and rats (Pagliarani et al., 2013). Evidence exists that TBT exposure tends to increase testosterone levels in female mollusks (Alzieu, 2000). In our study, HSD17B10 was found to be one of the top three signifi- cantly up-regulated proteins related to hormone homeostasis. As a member of the dehydrogenase/reductase superfamily, HSD17B10 plays critical roles in fatty acid oxidation, amino acid degradation and metabolism of sex hormones and neuroactive steroids (Yang et al., 2014). HSD17B10 has been demonstrated to be involved in the degra- dation of glucocorticoids and sex steroids and the epimerization of bile acids (Moeller and Adamski, 2009). Fig. S5A shows that HSD17B10 is involved in important cellular functions such as regulation of the NF-KB signaling pathway and developmental disorders and was the central molecule in the top scoring network.
TBT is considered to be an obesogenic compound, as well as an endo- crine disrupting compound (Casals-Casas and Desvergne, 2011). In this study, BTs were observed to significantly affect the protein expression levels of 3-ketoacyl-CoA thiolase (ACAA2), enoyl-CoA hydratase (ECHS1) and FABP5, which are directly involved in fatty acid metabo- lism. ECHS1 and ACAA2 are essential in the mitochondrial fatty acid beta-oxidation pathway. FABP5 is important in fatty acid uptake, trans- port, and metabolism. FABP5 has been reported to be downstream of the PPARy/RXR signaling and found to be upregulated in 3T3-L1 cells by TBT treatment (Pereira-Fernandes et al., 2013). The expression of the adipogenic markers FABP4, an isoform of FABP5, was significantly induced by TBT and DBT in 3T3-L1 cells (Milton et al., 2017). In the func- tionally enriched pathway analysis, fatty acid metabolism, fatty acid degradation and fatty acid elongation pathways were all affected by BTs, disrupting cellular fatty acid homeostasis. The alteration of related protein expression and significant enrichment of proteins in the fatty acid metabolism pathway revealed the obesogenic effects of BTs on
H295R cells. The biomagnification of BTs through marine food-webs may pose a potential threat to lipid metabolism in aquatic organisms and to human beings.
Renal necrosis/cell death was found to be one of the main or final targets of BTs. In our study, several proteins (LGALS7B, CD5L, SFN and NDRG1) involved in cell apoptosis were found to be affected by BTs. Pre- vious studies have also shown that TBT can cause apoptosis in several rat and human cell lines and the organs of rockfish (Mundy and Freudenrich, 2006; Jurkiewicz et al., 2004; Li et al., 2015; Zhang et al., 2011). TBT (1 µM) also induced morphologic changes in H295R cells with cell shrinkage and reduced adhesion (Fig. S2).
Except for the above mentioned pathways and targets, bioinformatic analyses revealed that BTs also affected the proteins involved with en- ergy metabolism, stress response, proteasomes, transport and a number of human diseases. Furthermore, the top networks show that the interacting proteins were part of the categories for metabolic diseases, organismal dysfunction and immunological diseases. The diseases and disorders resulting from the BTs-regulated proteins provide potential damage to human health.
5. Conclusion
We report here that BTs have complex actions, including endocrine disrupting effects, on H295R cells. Proteins related to hormone and lipid metabolism were significantly up- or down- regulated following treatment with BTs. Investigation of the proteome showed that BTs have the ability to activate nuclear receptor pathways, LXR/RXR and FXR/RXR, and thereby modulate cellular homeostasis. Our study re- vealed that TBT up-regulated the expression of LXRox and FXR mRNA, and confirmed the activation of LXR/RXR and FXR/RXR in H295R cells. Moreover, BTs disrupted a number of proteins related to basic cellular processes, such as, energy metabolism, stress response, and cell necro- sis. Overall, these findings indicate that BTs cause activation of LXR/ RXR and FXR/RXR and also have effects on sterol, glucose and lipid me- tabolism and offer valuable insights into their potential effects on humans. Nevertheless, further studies are needed to clarify the details in the nuclear receptor pathways to elucidate the complex actions of BTs in mammals in vitro and in vivo.
Acknowledgements
This study was supported by the National Nature Science Foundation of China (21277151, 21577153, 21777179 and 21537004) and The Thousand Talents Plan for Young Professionals, China.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.02.165.
References
Alzieu, C., 2000. Impact of tributyltin on marine invertebrates. Ecotoxicology 9 (1), 71-76. Antizar-Ladislao, B., 2008. Environmental levels, toxicity and human exposure to tributyl- tin (TBT)-contaminated marine environment. A review. Environ. Int. 34 (2), 292-308.
Bertuloso, B.D., Podratz, P.L., Merlo, E., Araújo, J.F.P., Lima, L.C.F., Miguel, E.C., Souza, L.N., Gava, A.L., Oliveira, M., Miranda-Alves, L., Carneiro, M.T.W.D., Nogueira, C.R., Graceli, J.B., 2015. Tributyltin chloride leads to adiposity and impairs metabolic functions in the rat liver and pancreas. Toxicol. Lett. 235, 45-59.
Busk, O.L., Ndossi, D., Verhaegen, S., Connolly, L., Eriksen, G., Ropstad, E., Sorlie, M., 2011. Relative quantification of the proteomic changes associated with the mycotoxin zearalenone in the H295R steroidogenesis model. Toxicon 58, 533-542.
Busk, O.L., Frizzell, C., Verhaegen, S., Uhlig, S., Connolly, L., Ropstad, E., Sorlie, M., 2012. Cy- tosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, alpha- and beta-zearalenol. Toxicon 59, 17-24.
Cai, S.Y., He, H.H., Nguyen, T., Mennone, A., Boyer, J.L., 2010. Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms. J. Lipid Res. 51 (8), 2265-2274.
Calkin, A.C., Tontonoz, P., 2012. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR. Nat. Rev. Mol. Cell Biol. 13, 213-224.
Cao, D.D., Jiang, G.B., Zhou, Q.F., Yang, R.Q., 2009. Organotin pollution in China: an over- view of the current state and potential health risk. J. Environ. Manag. 90, S16-S24.
Cao, S., Ye, L., Wu, Y., Mao, B., Chen, L., Wang, X., Huang, P., Su, Y., Ge, R .- S., 2017. The ef- fects of fungicides on human 3B-Hydroxysteroid dehydrogenase 1 and aromatase in human placental cell line JEG-3. Pharmacology 100, 139-147.
Casals-Casas, C., Desvergne, B., 2011. Endocrine disruptors: from endocrine to metabolic disruption. Annu. Rev. Physiol. 73, 135-162.
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.
Cima, F., Ballarin, L., 2012. Genotoxicity and immunotoxicity of organotins. In: Pagliarani, A., Trombetti, F., Ventrella, V. (Eds.), Biochemical and Biological Effects of Organotins, pp. 97-111.
Commendatore, M.G., Franco, M.A., Gomes Costa, P., Castro, I.B., Fillmann, G., Bigatti, G., Esteves, J.L., Nievas, M.L., 2015. Butyltins, polyaromatic hydrocarbons, organochlorine pesticides, and polychlorinated biphenyls in sediments and bivalve mollusks in a mid-latitude environment from the Patagonian coastal zone. Environ. Toxicol. Chem. 34 (12), 2750-2763.
Ferreira, M., Blanco, L., Garrido, A., Vieites, J.M., Cabado, A.G., 2013. In vitro approaches to evaluate toxicity induced by organotin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) in neuroblastoma cells. J. Agric. Food Chem. 61 (17), 4195-4203.
Gamberi, T., Magherini, F., Fiaschi, T., Landini, I., Massai, L., Valocchia, E., Bianchi, L., Bini, L., Gabbiani, C., Nobili, S., Mini, E., Messori, L., Modesti, A., 2015. Proteomic analysis of the cytotoxic effects induced by the organogold(III) complex Aubipy- in cisplatin- resistant A2780 ovarian cancer cells: further evidence for the glycolytic pathway im- plication. Mol. BioSyst. 11 (6), 1653-1667.
Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., Chrousos, G.P., Brennan, M.F., Stein, C.A., Larocca, R.V., 1990. Establishment and characterization of a human adrenocortical carcinoma cell-line that expresses multiple pathways of steroid-biosynthesis. Cancer Res. 50 (17), 5488-5496.
Görg, A., Postel, W., Gunther, S., 1988. The current state of two-dimensional electrophore- sis with immobilized pH gradients. Electrophoresis 9 (9), 531-546.
Görg, A., Postel, W., Friedrich, C., Kuick, R., Strahler, J.R., Hanash, S.M., 1991. Temperature- dependent spot positional variability in two-dimensional polypeptide patterns. Elec- trophoresis 12, 653-658.
Görg, A., Weiss, W., Dunn, M.J., 2004. Current two-dimensional electrophoresis technol- ogy for proteomics. Proteomics 4 (12), 3665-3685.
Graceli, J.B., Sena, G.C., Lopes, P.F., Zamprogno, G.C., da Costa, M.B., Godoi, A.F., Dos Santos, D.M., de Marchi, M.R., Dos Santos Fernandez, M.A., 2013. Organotins: a review of their reproductive toxicity, biochemistry, and environmental fate. Reprod. Toxicol. 36, 40-52.
Grün, F., Watanabe, H., Zamanian, Z., Maeda, L., Arima, K., Chubacha, R., Gardiner, D.M., Iguchi, T., Kanno, J., Blumberg, B., 2006. Endocrine disrupting organotin compounds are potent inducers of adipogenesis in vertebrates. Mol. Endocrinol. 20 (9), 2141-2155.
Hecker, M., Newsted, J.L., Murphy, M.B., Higley, E.B., Jones, P.D., Wu, R., Giesy, J.P., 2006. Human adrenocarcinoma (H295R) cells for rapid in vitro determination of effects on steroidogenesis: hormone production. Toxicol. Appl. Pharmacol. 217 (1), 114-124.
Hecker, M., Hollert, H., Cooper, R., Vinggaard, A.M., Akahori, Y., Murphy, M., Nellemann, C., Higley, E., Newsted, J., Laskey, J., Buckalew, A., Grund, S., Maletz, S., Giesy, J., Timm, G., 2011. The OECD validation program of the H295R steroidogenesis assay: phase 3. Final inter-laboratory validation study. Environ. Sci. Pollut. Res. 18, 503-515.
Ho, K.K.Y., Leung, K.M.Y., 2014. Organotin contamination in seafood and its implication for human health risk in Hong Kong. Mar. Pollut. Bull. 85 (2), 634-640.
Hollert, H., Giesy, J., 2007. The OECD validation program of the H295R steroidogenesis assay for the identification of in vitro inhibitors and inducers of testosterone and es- tradiol production. Phase 2: inter-laboratory pre-validation studies (8 pp). Environ. Sci. Pollut. Res. 14 (Suppl. 1), 23-30.
Jiang, G.B., Zhou, Q.F., Liu, J.Y., Wu, D.J., 2001. Occurrence of butyltin compounds in the waters of selected lakes, rivers and coastal environments from China. Environ. Pollut. 115 (1), 81-87.
Jurkiewicz, M., Averill-Bates, D.A., Marion, M., Denizeau, F., 2004. Involvement of mito- chondrial and death receptor pathways in tributyltin-induced apoptosis in rat hepa- tocytes. Biochim. Biophys. Acta 1693 (1), 15-27.
Kanayama, T., Kobayashi, N., Mamiya, S., Nakanishi, T., Nishikawa, J., 2005. Organotin compounds promote adipocyte differentiation as agonists of the peroxisome roliferator-activated receptor gamma/retinoid X receptor pathway. Mol. Pharmacol. 67 (3), 766-774.
Kannan, K., Senthilkumar, K., Giesy, J.P., 1999. Occurrence of butyltin compounds in human blood. Environ. Sci. Technol. 33 (10), 1776-1779.
Lardinois, O., Kirby, P.J., Morgan, D.L., Sills, R.C., Tomer, K.B., Deterding, L.J., 2014. Mass spectrometric analysis of rat cerebrospinal fluid proteins following exposure to the neurotoxicant carbonyl sulfide. Rapid Commun. Mass Spectrom. 28, 2531-2538.
Lawrence, S., Reid, J., Whalen, M., 2015. Secretion of interferon gamma from human im- mune cells is altered by exposure to tributyltin and dibutyltin. Environ. Toxicol. 30 (5), 559-571.
Leung, K.M.Y., Kwong, R.P.Y., Ng, W.C., Horiguchi, T., Qiu, J.W., Yang, R.Q., Song, M.Y., Jiang, G.B., Zheng, G.J., Lam, P.K.S., 2006. Ecological risk assessments of endocrine disrupting organotin compounds using marine neogastropods in Hong Kong. Chemosphere 65 (6), 922-938.
Li, B., Sun, L., Cai, J., Wang, C., Wang, M., Qiu, H., Zuo, Z., 2015. Modulation of the DNA re- pair system and ATR-p53 mediated apoptosis is relevant for tributyltin-induced genotoxic effects in human hepatoma G2 cells. J. Environ. Sci. 27 (1), 108-114.
Liu, Z., Lin, Y., Zhang, S., Wang, D., Liang, Q., Luo, G., 2015. Comparative proteomic analysis using 2DE-LC-MS/MS reveals the mechanism of Fuzhuan brick tea extract against he- patic fat accumulation in rats with nonalcoholic fatty liver disease. Electrophoresis 36, 2002-2016.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402-408. Milton, F.A., Lacerda, M.G., Sinoti, S.B.P., Mesquita, P.G., Prakasan, D., Coelho, M.S., de Lima, C.L., Martini, A.G., Pazzine, G.T., Borin, M.D., Amato, A.A., Neves, F.D.R., 2017. Dibutyltin compounds effects on PPARy/RXRc activity, adipogenesis, and inflamma- tion in mammalians cells. Front. Pharmacol. 8, 507.
Mitra, S., Gera, R., Siddiqui, W.A., Khandelwal, S., 2013. Tributyltin induces oxidative dam- age, inflammation and apoptosis via disturbance in blood-brain barrier and metal ho- meostasis in cerebral cortex of rat brain: an in vivo and in vitro study. Toxicology 310 (9), 39-52.
Mitra, S., Siddiqui, W.A., Khandelwal, S., 2015. Differential susceptibility of brain regions to tributyltin chloride toxicity. Environ. Toxicol. 30 (12), 1393-1405.
Moeller, G., Adamski, J., 2009. Integrated view on 17beta-hydroxysteroid dehydroge- nases. Mol. Cell. Endocrinol. 301 (1-2), 7-19.
Mundy, W.R., Freudenrich, T.M., 2006. Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: involvement of MAP kinases. Neurotoxicology 27 (1), 71-81.
Nakanishi, T., 2008. Endocrine disruption induced by organotin compounds; organotins function as powerful agonists for nuclear receptors rather than an aromatase inhibi- tor. J. Toxicol. Sci. 33 (3), 269-276.
Nishikawa, J.I., Mamiya, S., Kanayama, T., Nishikawa, T., Shiraishi, F., Horiguchi, T., 2004. Involvement of the retinoid X receptor in the development of imposex caused by organotins in gastropods. Environ. Sci. Technol. 38 (23), 6271-6276.
Oberdõrster, E., McClellan-Green, P., 2002. Mechanisms of imposex induction in the mud snail Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor. Mar. Environ. Res. 54, 715-718.
Oberemm, A., Hansen, U., Bömert, L., Meckert, C., Braeuning, A., Thüemann, A.F., Lampen, A., 2016. Proteomic responses of human intestinal Caco-2 cells exposed to silver nanoparticles and ionic silver. J. Appl. Toxicol. 36 (3), 404-413.
Omura, M., Ogata, R., Kubo, K., Shimasaki, Y., Aou, S., Oshima, Y., Tanaka, A., Hirata, M., Makita, Y., Inoue, N., 2001. Two-generation reproductive toxicity study of tributyltin chloride in male rats. Toxicol. Sci. 64 (2), 224-232.
Pagliarani, A., Nesci, S., Ventrella, V., 2013. Toxicity of organotin compounds: shared and unshared biochemical targets and mechanisms in animal cells. Toxicol. In Vitro 27 (2), 978-990.
Pandey, A., Mann, M., 2000. Proteomics to study genes and genomes. Nature 405, 837-846.
Pereira-Fernandes, A., Vanparys, C., Hectors, T.L.M., Vergauwen, L., Knapen, D., Jorens, P.G., Blust, R., 2013. Unraveling the mode of action of an obesogen: mechanistic analysis of the model obesogen tributyltin in the 3T3-L1 cell line. Mol. Cell. Endocrinol. 370 (1-2), 52-64.
Podratz, P.L., Merlo, E., Sena, G.C., Morozesk, M., Bonomo, M.M., Matsumoto, S.T., Da Costa, M.B., Zamprogno, G.C., Brandão, P.A., Carneiro, M.T., Miguel, E.C., Miranda-Alves, L., Silva, I.V., Graceli, J.B., 2015. Accumulation of organotins in seafood leads to reproduc- tive tract abnormalities in female rats. Reprod. Toxicol. 57, 29-42.
Sanderson, J.T., 2006. The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals. Toxicol. Sci. 94 (1), 3-21.
Sanderson, J.T., Boerma, J., Lansbergen, G.W.A., van den Berg, M., 2002. Induction and in- hibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. 182, 44-54.
Sena, G.C., Freitas-Lima, L.C., Merlo, E., Podratz, P.L., de Araújo, J.F.P., Brandão, P.A.A., Carneiro, M.T.W.D., Zicker, M.C., Ferreira, A.V.M., Takiya, C.M., Barbosa, C.M.D., Mo- rales, M.M., Santos-Silva, A.P., Miranda-Alves, L., Silva, I.V., Graceli, J.B., 2017. Environ- mental obesogen tributyltin chloride leads to abnormal hypothalamic-pituitary- gonadal axis function by disruption in kisspeptin/leptin signaling in female rats. Toxicol. Appl. Pharmacol. 319, 22-38.
Shi, X., Yeung, L.W., Lam, P.K., Zhou, B., 2009. Protein profiles in zebrafish (Danio rerio) embryos exposed to perfluorooctane sulfonate. Toxicol. Sci. 110, 334-340.
Tang, H., Mirshahidi, S., Senthil, M., Kazanjian, K., Chen, C.S., Zhang, K.L., 2014. Down- regulation of LXR/RXR activation and negative acute phase response pathways in colon adenocarcinoma revealed by proteomics and bioinformatics analysis. Cancer Biomark 14 (5), 313-324.
Yang, S.Y., He, X.Y., Isaacs, C., Dobkin, C., Miller, D., Philipp, M., 2014. Roles of 17ß- hydroxysteroid dehydrogenase type 10 in neurodegenerative disorders. J. Steroid Biochem. Mol. Biol. 143, 460-472.
Zhang, J., Zuo, Z., Wang, Y., Yu, A., Chen, Y., Wang, C., 2011. Tributyltin chloride results in dorsal curvature in embryo development of Sebastiscus marmoratus via apoptosis pathway. Chemosphere 82 (3), 437-442.
Zhang, J., Zuo, Z., Xiong, J., Sun, P., Chen, Y., Wang, C., 2013. Tributyltin exposure causes lipotoxicity responses in the ovaries of rockfish, Sebastiscus marmoratus. Chemosphere 90 (3), 1294-1299.
Zhou, Q.F., Jiang, G.B., Liu, J.Y., 2002. Effects of sublethal levels of tributyltin chloride in a new toxicity test organism: the Chinese rare minnow (Gobiocypris rarus). Arch. Envi- ron. Contam. Toxicol. 42 (3), 332-337.