- Email: [email protected]
Understanding the interaction of single-walled carbon nanotube (SWCNT) on estrogen receptor: A combined molecular dynamics and experimental study
Ecotoxicology and Environmental Safety 172 (2019) 373–379
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Understanding the interaction of single-walled carbon nanotube (SWCNT) on estrogen receptor: A combined molecular dynamics and experimental study
T
Xinhe Liua, Tingting Liub, Juanjuan Songc, Ying Haia, Feng Luand, Haixia Zhange, Yongna Yuanf, ⁎ Hongyu Lia, Chunyan Zhaoa, a
School of Pharmacy, Lanzhou University, Lanzhou 730000, China Gansu Provincial Maternity and Child-care Hospital, Lanzhou 730000, China Pulmonary Hospital of Lanzhou, Lanzhou 730000, China d College of Chemistry and Chemical Engineering, Yantai University, Yantai 264000, China e College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China f School of Information Science & Engineering, Lanzhou University, Lanzhou, 730000, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Single-walled carbon nanotube (SWCNT) Estrogen receptor (ER) Molecular dynamics Fluorescence
Considering the large-scale production of diversified nanomaterials, it is paramount importance to unravel the structural details of interactions between nanoparticles and biological systems, and thus to explore the potential adverse impacts of nanoparticles. Estrogen receptors (ER) is one of the most important receptor of human reproductive system and the binding of carbon nanotubes to estrogen receptors was the possible trigger leading to the reproductive toxicity of carbon nanotubes. Thus, with single-walled carbon nanotube (SWCNT) treated as model nanomaterials, a combination of in vivo experiments, spectroscopy assay and molecular dynamic modeling was applied to help us unravel some important issues on the binding characterization between SWCNT and the ligand binding domain (LBD) of ER alpha (ERα). The fluorescence assay and molecular dynamics simulations together validated the binding of SWCNT to ERα, suggesting the possible molecular initiating event. As a consequence, SWCNT binding led to a conformational change on tertiary structure levels and hydrophobic interaction was recognized as the driving force governing the binding behavior between SWCNT and LBD of ERα. A in vivo process presented that the exposure of SWCNT increased ERα expression from 26.43 pg/ml to 259.01 pg/ml, suggesting a potential estrogen interference effects of SWCNT. Our study offers insight on the binding of SWCNT and ERα LBD at atomic level, helpful to accurately evaluate the potential health risks of SWCNT.
1. Introduction The number of nanomaterials products has been keeping increasing over the past decade (Guo et al., 2017; Wang et al., 2018). Nanoparticles also have great potential in biomedical applications such as drug delivery, radiation therapy, chemotherapy and biosensors (Gaunt et al., 2015; Matsumoto et al., 2015; Ray et al., 2018). However, the biological effects of nanomaterials are extremely unpredictable (Suh et al., 2009). In particular, it can interact with biological macromolecules. Therefore, proteins have always been a research focus in the interaction between nanomaterials and biomacromolecules (Chang et al., 2017; Leal et al., 2015; Yue and Zhang, 2012). For example, it is reported that fullerene (C60) and single-walled carbon nanotubes
⁎
(SWCNT) can effectively interacting with the hydrophobic binding sites on HIV-1 protease (HIV-1P)] and glutathione-S-transferase (GST), which leads to a suppression the activity of enzymes (Iwata et al., 1998; Karelson and Martin, 2010; Zhu et al., 2003). On the other sides, an injected nanoparticle into a living system could also result in an uncountable number of interactions (Beddoes et al., 2015; Oner et al., 2018). The highly unintended or undesired interactions of nanomaterials with biological molecules will cause side effects with a strong possibility, which thus may induce so-called cytotoxicity. Now, more and more nanotoxicology researches have been focused on the understanding the details of unintended or undesired interactions of nanomaterials with biological molecule (Chen et al., 2015; Chortarea et al., 2017; Rancan et al., 2012). Growing number of studies have proved
Corresponding author. E-mail address: [email protected] (C. Zhao).
https://doi.org/10.1016/j.ecoenv.2019.01.101 Received 30 October 2018; Received in revised form 21 January 2019; Accepted 29 January 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
2.2. Fluorescence measurement
that NPs can cause adverse physiological effects such as inflammation and immunological responses that lead to dysfunction of the tissues and organs. For example, Zuo et al. summarized the interactions between proteins and carbon nanotubes (CNTs), which should be a necessary step to understand the molecular mechanisms of nanotoxicity. At present, as the most commonly used nanomaterial, carbon nanotubes have recently been reported to have reproductive toxicity (Xu et al., 2017; Zhang et al., 2010). It has been reported that intravenous injection of carbon nanotubes into adult female mice causes transient histopathological changes (Wang et al., 2014). Intraperitoneal injection of multiwalled nanotubes in mice can cause malformations, miscarriage and can also increase the serum estradiol levels (Campagnolo et al., 2013; Ryoo et al., 2010). Estrogen receptors is one of the important parts of human reproductive system, the interaction between carbon nanotubes and estrogen receptors may be one of the important reasons for the reproductive toxicity of carbon nanotubes (Bisesi et al., 2017; Johansson et al., 2017). As ligand-regulated transcription factors, the biological effects of estrogen receptors are mediated primarily by binding to the endogenous estrogen estradiol-17β (E2) (Acaz-Fonseca et al., 2014; Kamanga-Sollo et al., 2017). However, many structurally diverse exogenous compounds, including phytochemicals, hydroxylated polychlorinated biphenyls, pesticides and other industrial compounds have been reported to bind to estrogen receptors and prevent unexpected estrogen-like activities (Collins-Burow et al., 2012; Pestana et al., 2015; Zhang et al., 2018). It may be one of the reasons that affects the reproductive system and leads to reproductive toxicity. For carbon nanotubes, given that it is structurally compatible with estrogen receptors, we hypothesized that, similar to other exogenous chemicals, carbon nanotubes can also interact with estrogen receptors to activate estrogen receptors and affect the reproductive system. However, the details of the interaction between carbon nanotubes and estrogen receptors are still unclear. In fact, an understanding of the mechanisms of estrogen receptor and carbon nanotubes binding may be a key stage in exploring how reproductive toxicity occurs and predicting the potential for adverse biological effects. Therefore, in this study, three methods were applied, including molecular dynamics simulations, fluorescence experiments and animal experiments to reveal the structural properties and details of the interaction between single-walled nanotubes and estrogen receptors. Given that evaluating the potential adverse biological effects of nanomaterials is much more complex, computer-based methods have been constructed that can help us understand the mechanisms of action of toxicity and the molecular details of their interactions with biomolecules. Due to the wide application of carbon nanotube materials, it is particularly important to study the structural characteristics and details of the interaction between nanomaterials and biological systems at the molecular level, thus further predicting and evaluating the potential toxicological effects of SWCNTs.
The instrument used in the fluorescence titration experiment was Model RF-5301 (Shimadzu Corporation, Japan). The fluorescence excitation wavelength was set to 385 nm and the slit width was set to λem = 5 nm and λex = 5 nm. Coumestrol (CS) was selected as the fluorescence spectroscopy probe targeting estrogen receptor. Concentration of ERα solution was fixed at 0.8 μM (2.64 × 10−2 g/L) with PBS buffer of 0.1 M and PH = 7. A targeted estrogen receptor fluorescent probe was added at a concentration of 0.1 μM (2.68 × 10−5 g/L) CS. The SWCNT suspension was gradually titrated to a final concentration of 0, 10−4 g/L, 2 × 10−4 g/L, 4 × 10−4 g/L, 6 × 10−4 g/L, 8 × 10−4 g/L. Fluorescence intensity was then recorded at a fluorescence excitation wavelength of 385 nm with a time interval of ten minutes (Han et al., 2013; Zhang et al., 2014; Zhuang et al., 2016). 2.3. Molecular dynamics The protein sequence of Rattus norvegicus ERα (rERα) was retrieved from the NCBI (http://www.ncbi.nlm.nih.gov) with sequence number of BAI48013. Homology models of rERα ligand-binding domain were constructed using Swiss-model with human ERα (hER PDB ID: 1GWR) as the template. More information about homology modeling presented in Roy et al. (2010). The identified best model was based on highest C-score, which was obtained from the relative clustering structural density and consensus significance. Besides it, Ramachandran plots calculated by Rampage program were used to explore the stereo chemical quality of the constructed ERα models. The initial 3D structures of rERα obtained from homology modelings were refined by 1 ns MD simulation with FF03 force field. The structure of the single-walled nanotubes was constructed using the Nanotube Builder module in the VMD software. The chirality m = n = 5 and the length was set to 1.0 nm. The constructed single-walled nanotubes contained 100 carbon atoms in total. Docking of singlewalled nanotubes to constructed rERα was performed using Autodock software 4.2.5.1. Molecular dynamics simulation of 50 ns was then applied to the SWCNT-ERα system using Amber14, in which the ERα protein was processed using FF03 force field and SWCNT was processed using GAFF force field. The module Tleap was used to hydrogenate the composite SWCNT-ERα system, which was then dissolved into a TIP3P periodic regular hexahedral water box to set any atom in the composite SWCNT-ERα. The minimum distance of the water box boundary is a minimum of 5 Å. The water molecules in the solution are replaced with positively charged sodium ions to make the entire system electrically neutral. All key lengths are limited by the SHAKE algorithm. The Verlet Frog Leaping Algorithm is used to set the integration step in the whole process of the dynamics to 2 fs. The limiting force for setting the composite SWCNT-ERα was set to 100 kcal/mol Å2 for water. The optimization of molecules and some counter particles was first performed using the steepest descent method of 3000 steps followed by a 2000 steps conjugate gradient method. The whole system was then optimized using the first method without any restriction. Subsequently, the temperature of the entire system was increased from 0 K to 298 K within 50 ps. Then a 100 ns dynamic simulation was performed at a 298 K NPT system (Ding et al., 2017; Lu et al., 2018; Shulin et al., 2014). The average structures derived from the last 30 ns trajectory of ERαSWCNT system were extracted for the further analysis. The Ramachandran plot was performed by the Rampage program. The distance and contact area were calculated using the Chimera 1.13.1. The residue-residue interaction networks (RIN) analysis was performed by Cytoscape 3.4.0 and the plugin RINalyzer. The binding energy of SWCNT-ERα system was calculated by the method about MM-GBSA. The Root Mean Square Deviation (RMSD) and Root-mean-square fluctuation (RMSF) analyses were performed by Origin8. The interhelical angles of H12 was calculated using the g_angle procedure in GROMACS 5.0 software.
2. Materials and methods 2.1. Chemicals and test solutions The Single-walled nanotubes solutions (1 g/L) were purchased from Shenzhen Nangang Port. Co., Ltd. (Shenzhen, China). The estrogen receptor fluorescent probe Coumestrol (CS) was purchased from J&K Blingway Technology Co., Ltd. Elisa kit (Rat Estradiol, E2 ELISA Kit) was purchased from Wuhan Liuhe Biotechnology Co., Ltd. The estrogen recombinant proteins (200 μg/ml) were purchased from Shanghai Xinyu Co., Ltd., containing recombinant ERα ligand binding domain (LBD) protein ERα of amino acids 178–483 of rat ERα (rERα248–484) with two N-terminal tags. The SD (Sprague Dawley) rats, aged 2–3 weeks, were purchased from the Animal Experimental Center of Lanzhou University. 374
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
2.4. In vivo experimental procedures Twenty-four female SD rats with rat age 3–4 weeks and the weight at 180 ± 20 g rats were divided into three groups, including singlewalled nanotubes (SWCNT) group, estradiol (E2) group and blank control group. After one week of acclimatization, the eight rats of SWCNT group were intragastrically administered as a single-walled nanotube suspension at a concentration of 15 mg/kg/d. The eight rats of E2 group were intragastrically administered as estradiol solution at 200 μg/kg/d and the eight rats in blank control group were administered as normal saline. Fig. 1. The fluorescence of ERα (0.8 μM) and SWCNT (1–6) at concentrations of 0 (only ERα), 10−4 g/L, 2 × 10−4 g/L, 4 × 10−4 g/L, 6 × 10−4 g/L, 8 × 10−4 g/L. The scanning wavelength ranged from 300 nm to 500 nm, the excitation wavelength was 385 nm, and the measurement temperature was 298 K.
2.4.1. Serum estradiol levels measurement After four weeks of gavage, the eyeballs of the above 24 SD rats were bled and centrifuged. The serum was collected and the serum estradiol levels were tested using the Elisa kit (Rat Estradiol, E2 ELISA Kit) (Qu et al., 2016).
residues in favored regions, 1.3% residues in allowed regions, and 0.4% residues in outlier regions. Ramachandran plots proved that the constructed rERα model was reasonable. The binding site of hERα was defined as the residues around 9 Å distance of the ligand estradiol. Considering that the similarity of rERα and hERα proteins, the binding sites of rERα were characterized as the same regions. Docking of singlewalled nanotubes to constructed rERα was performed. Molecular dynamic (MD) simulations were further used to explore the biological processes between the SWCNT and the ERα. The stability of the entire complex system was analyzed by root-mean-square deviations (RMSD) analysis (Fig. S2). The RMSD was calculated by extracting the relevant data from the trajectory file. It can be seen that the ERα-SWCNT system has reached basic stability with no significant fluctuations after 40 ns. The average structures derived from the last 20 ns trajectory of ERα-SWCNT system were then extracted for the further analysis.
2.4.2. Western blot As shown, the SD rats were sacrificed by spinal dislocation. The uterine tissues were taken and stored in a cryogenic freezer at −70 °C. The tissue sample was added into cold Lysis buffer and centrifuged. The supernatant was taken as a whole protein extract and protein ration was tested using Bradford method. The extracted protein was then transferred to a nitrocellulose (NC) filter. Membranes were blocked with blocking buffer (5% nonfat dry milk in Tween-20 (TBST) Tris buffered saline) and incubated with TBST after 30 min of incubation at room temperature. The membranes were then incubated against primary antibody of ERα rabbit polyclonal antibody at 4 °C overnight. After overnight incubation, the membranes were washed three times at room temperature with TBST and then incubated with a biotinylated secondary antibody (rabbit anti-GAPDH (10B8)) for 2 h at room temperature. The secondary antibody was then washed with TBST and the color was imaged. The imaging was performed using G:BOX chemiXR5 software and gray-scale analysis using Gel-Pro32 software. The expression of glycoaldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control and the density ratio of protein density to GAPDH for grayscale analysis were calculated (Yao et al., 2014; Zhan et al., 2018). The statistical analysis of Estradiol (E2) level assay was applied using the SPSS 11.5 statistical software package, with value of p < 0.05 defined as significance. Parametric comparison was performed by one-way Analysis of Variance (ANOVA) followed by Dunnett-t-test.
3.2.2. Binding modes of SWCNT to ERα Molecular dynamics (MD) simulations were used to explore the model dynamic fluctuations in the structure of ERα upon binding to the single-wall carbon nanotube. This binding process between SWCNT and ERα can be illustrated by the interface area between SWCNT and the ER protein domain (Fig. 2A). The parameter of interface area S was defined as half of the difference between the solvent-accessible surface area of the complex and the sum of solvent-accessible surface areas of the protein-SWCNT complex. The important SWCNT-ERα complex snapshots at different times were displayed to present the process of SWCNT plugging into ER. At the beginning, the value of interface area S presented a very small value of 334 Å2. The area S increased very quickly to 340 Å2 within the first 5 ns, presenting that binding sites of ER and the SWCNT approached each other very quickly. The area S slowly increased from 340 to 345 Å2 in the next 10 ns. The area S increased reached its maximal value of 354 Å2 at 25 ns. After 35 ns, it is basically
3. Result and discussion 3.1. Fluorescence analysis for SWCNT-ERα LBD interaction Fluorescence titration experiment is an effective method to explore the effects of single-walled nanotubes (SWCNT) on the structural changes of LBD of ERα. As shown, fluorescence spectrum of the estrogen receptor was recorded in the presence of SWCNTs (Fig. 1). Fluorescent probe CS was added to increase the fluorescence intensity of the estrogen receptor and the fluorescence emission spectra were measured as the average of three scans at ten minute intervals. As presented, as the concentrations of SWCNT increasing, the fluorescence intensity of the ERα gradually decreased. The strong fluorescence quenching clearly showed the interaction between the SWCNT and the estrogen receptor. It also reflected that tertiary structures of ERα have changed due to the addition of SWCNTs.
Fig. 2. (A) The SWCNT-ERα interface areas as a function of time. Some representative snapshots (5 ns, 15 ns, 25 ns and 35 ns) were also presented. SWCNT was presented as gold and rERα was presented as blue. (B) Binding modes of SWCNT in the binding pocked of ERα. Structures of ERα were presented as ribbon. The blue ribbon represented free bound ERα protein and the golden ribbon represented SWCNT-ERα.
3.2. Molecular dynamics modeling 3.2.1. Homology modeling The constructed homology model of rERα was examined by the Ramachandran plot (Fig. S1). The rERα model presented 98.3% 375
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
were all located near the binding cavity. The above 15 amino acids can further be classified as polar amino acids and non-polar amino acids. Among the above 15 amino acids that contributed greatly, there were only 2 polar amino acids, but 13 non-polar amino acids, accounting for 86.7% of the total. As known, the polar amino acids have strong hydrophilicity, while the nonpolar polar amino acids have strong hydrophobicity. Therefore, it can be seen (Fig. 3), that the contribution of hydrophobic interaction was the main force of binding behavior, while electrostatic interaction contributed very little and almost no contribution. Based on these results, it can be concluded that hydrophobic interactions can be attributed to the driving forces that play an important role in ERα-SWCNT binding.
straight, indicating that the contact surface of the domain has reached a relatively stable state. It suggested that SWCNT was finally successfully inserted into the bonding cavity of rERα and remained stable.
3.2.3. Binding free energy and energy decomposition In order to judge the binding ability of single-walled nanotubes (SWCNT) to ERα, MMGBSA method was used to calculate the binding free energy of the ERα-SWCNT system, which presented as ΔGtotal = −34.51 kcal/mol in Table S1 (Supporting information). The total free energy can be decomposed into its components as shown in Table S1. The molecular mechanics electrostatic contribution (ΔEele ) was counterbalanced by the contribution of electrostatic of the solvation energy (ΔEgb ). A value of 7.21 kcal/mol showing that the total electrostatic contribution (ΔEgb + ΔEele ) was slightly non-favorable for the binding. The van derWaals contribution together with the non-polar solvation free energy (ΔE vdws + ΔEsurf ) constituted the non-polar energy term and a value of −41.72 kcal/mol presented a significant favorable contribution to the overall binding free energy. As known, the total binding free energy value was more negative and the combination of between SWCNT and ERα was better. Here, the relatively low value of the total energy suggested that the binding affinity between SWCNT and ERα was quite strong and the binding was stable. The result of the total energy analysis was also consistent with the fluorescence titration experiment, demonstrating a strong combination of SWCNT to the ligand binding domain of ERα. In addition, the negative value of ΔE vdws indicates that the hydrophobic interaction between SWCNT and ERα promoted favorable binding. The highly favorable non-polar component originated from the van derWaals energy, which also demonstrated hydrophobic interactions as a key force governing the bindings between SWCNT and ERα. Besides it, energy contribution difference analysis was performed to rationalize the relative importance of contributions for each residue (Fig. 3). It was found that there were 15 amino acids that contributed a lot, namely Ser214, His220, Leu42, Leu80, Leu83, Leu87, Leu124, Leu221, Met117, Met213, Met218, Ile120, Phe121 and Gly217, which
3.2.4. Residue interaction network The residue interaction network (RIN) is a new technology to identify key amino acid interactions and to identify the topological structure of the entire system (Fig. 4). RIN is a modern topology based analysis which helped in identification of residue–residue contact difference in biological systems, where the nodes represent amino acid residues and the edges represent the interactions between the residues. The structure of the ERα was extracted from the two system dynamics of SWCNT binding ERα (SWCNT- ERα) system (Fig. 4A) and the free bound ERα (Free-ERα) (Fig. 4B), respectively. As shown, in the free bound ERα, the total amino acid-amino acid interactions were 551, while in the SWCNT-ERα system, the total number of interactions was reduced to 497 with a reduction of 9.1%. The results presented that the binding of SWCNTs relaxed the structure of the protein. In order to further analyze the interactions between amino acids, we selected all the amino acids in the surrounding 5 Å range of the binding pocket, with a total of 28 amino acids, as shown in (Fig. 5). For the free bound ERα system, the total edges were 50, of which 20 corresponded to van derWaals contacts, 1 to ionic interactions and 29 to hydrogen bonds. While, in the SWCNT-ERα system, the total edges was reduced to 26, the hydrogen bonds was 5, the van derWaals force was 21 and the ionic interaction was 0, which showed the total decreasing rate of 48%, the hydrogen bonds decreasing rate of 75.0% and the van derWaals forces decreasing rate of 27.6%. In summary, the combination of SWCNTs severely damaged the mutual network of ERα residues, especially to hydrogen bonds, which resulted in the loosening of the overall ERα structure and the destruction of the secondary and tertiary structure of the protein. 3.2.5. H12 rotations analysis The root mean square fluctuations (RMSF) plot for the helix 12 residues and the surrounding residues was presented (Fig. S3). As seen, the residues in the region Helix 12 (228–236) showed higher fluctuations compared to the adjacent regions. One possible reason is that, H12, as the most important helix of ERα, its position had changed during the simulation, indicating SWCNT had already modified the conformation of H12. Thus, the movement and rotations of H12 were further explored because the position of H12 in the LBD of ERαs is the key factor governing the relative recruitment of coregulators to coordinate a transcriptional response (Warnmark et al., 2002). The structures of ERα complexed with agonist and antagonist ligands are induced into different conformations, particularly the position and orientation of H12 (Fig. 6A). The relative rotational orientations of H12 and interhelical angles were quantitatively examined in (Fig. 6B). As shown, the H12 in ERα with binding of SWCNT turned from free bound state to agonist state with an interhelical angle of 10.2°, which suggested that SWCNT presented an agonist effect in rat ERα. The analysis of the movement and rotational angle of Helix 12 indicated that SWCNT had agonist effect on rat ERα, which might lead to a further influence on the recruitment of coregulators and the subsequent transcriptional regulation.
Fig. 3. Binding energy analysis of SWCNT-ERα system. (A) Contribution of each amino acid to SWCNT and ERα. (B) Polarity (dark grey) and non-polar contribution (light gray) of key amino acids to binding freedom. Negative values are favorable for the combination of SWCNTs. 376
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
Fig. 4. Characterization of the residue-residue networks for the ERα residues of (A) Free bound ERα and (B) SWCNT-ERα system. SWCNT was presented using gold color. The red spheres presented residues of ERα. Lines indicated residue-residue interactions, including red lines present hydrogen bonds and blue lines present van derWaals force.
3.3. In vivo analysis 3.3.1. Western blot analysis To further analyze the effect of SWCNTs on the expression of estrogen receptor in rats, Western Blot immunoblot analysis was performed to determine the effect of SWCNTs on the expression of ERα in rat uterus. As can be seen (Fig. S4), the expression of ERα in the uterus of the rats administered E2 was slightly up-regulated compared with the control group, while the expression of ERα in the uterus of the rats treated with SWCNT alone was significantly down-regulated. This indicates that the addition of SWCNT significantly reduced the expression of ERα in the rat uterus. This also reflects the decrease in estrogen levels in the rat uterus after the addition of SWCNTs. This may cause changes in hormone levels in the uterus.
3.3.2. Estradiol (E2) level assay As known, estradiol (E2) level in serum is an important indicator to evaluate reproductive endocrine interference effect. To examine the effects of single-walled nanotubes on the reproductive system, estradiol in rat serum was measured. As shown in (Fig. 7), the level of estradiol in the blank control group was 26.43 pg/ml, followed by 259.01 pg/ml in the SWCNT group and 690.48 pg/ml in E2 group. The level of estradiol in the rat serum administered with the SWCNT group was much higher than that of the blank control group. It indicated that the addition of the SWCNTs raised the level of estradiol in the rats. Estradiol is an extremely important hormone in the body. Changes in estradiol levels in the body may be related to hormone feedback and pituitary regulation,
Fig. 6. (A) Positions of the H12 (blue ribbon) in free bound ERα system, agonist-bound and antagonist-bound state. The ER LBD was presented as red ribbon in free bound state and presented as electrostatic surface in agonist-bound and antagonist-bound state. (B) Interhelical angles of H12 of free bound ERα system and SWCNT-ERα complex. The interhelical angles were defined as the rotational angle of H12 from free bound state (blue color) to agonist state (golden color).
Fig. 5. Characterization of the residue-residue networks for the ERα residues surrounding 5 Å range of the binding pocket from (A) Free bound ERα; (B) SWCNT-ERα system. The yellow spheres presented residues of ERα. SWCNT was presented using gold color. Red lines indicate hydrogen bonds. Blue lines indicated van derWaals force and gray lines indicated ion interactions.
377
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
toxicity of pegylated single wall carbon nanotubes in pregnant mice. Part. Fibre Toxicol. 10, 21. Chang, L.F., He, X.W., Chen, L.X., Zhang, Y.K., 2017. A novel fluorescent turn-on biosensor based on QDs@GSH-GO fluorescence resonance energy transfer for sensitive glutathione S-transferase sensing and cellular imaging. Nanoscale 9, 3881–3888. Chen, S.M., Xiong, C.Q., Liu, H.H., Wan, Q.Q., Hou, J., He, Q., Badu-Tawiah, A., Nie, Z.X., 2015. Mass spectrometry imaging reveals the sub-organ distribution of carbon nanomaterials. Nat. Nanotechnol. 10, 176–182. Chortarea, S., Barosova, H., Clift, M.J.D., Wick, P., Petri-Fink, A., Rothen-Rutishauser, B., 2017. Human asthmatic bronchial cells are more susceptible to subchronic repeated exposures of aerosolized carbon nanotubes at occupationally relevant doses than healthy cells. Acs Nano 11, 7615–7625. Collins-Burow, B.M., Antoon, J.W., Frigo, D.E., Elliott, S., Weldon, C.B., Boue, S.M., Beckman, B.S., Curiel, T.J., Alam, J., McLachlan, J.A., Burow, M.E., 2012. Antiestrogenic activity of flavonoid phytochemicals mediated via the c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha. J. Steroid Biochem. Mol. Biol. 132, 186–193. Ding, K.K., Kong, X.T., Wang, J.P., Lu, L.P., Zhou, W.F., Zhan, T.J., Zhang, C.L., Zhuang, S.L., 2017. Side chains of parabens modulate antiandrogenic activity: in vitro and molecular docking studies. Environ. Sci. Technol. 51, 6452–6460. Gaunt, N.P., Patil-Sen, Y., Baker, M.J., Kulkarni, C.V., 2015. Carbon nanotubes for stabilization of nanostructured lipid particles. Nanoscale 7, 1090–1095. Guo, Q., You, H.H., Yang, X., Lin, B.C., Zhu, Z.H., Lu, Z.S., Li, X.X., Zhao, Y., Mao, L., Shen, S.P., Cheng, H.Y., Zhang, J.T., Deng, L.F., Fan, J.H., Xi, Z.G., Li, R., Li, C.M., 2017. Functional single-walled carbon nanotubes 'CAR' for targeting dopamine delivery into the brain of parkinsonian mice. Nanoscale 9, 10832–10845. Han, C., Fang, S.B., Cao, H.M., Lu, Y., Ma, Y.Q., Wei, D.F., Xie, X.Y., Liu, X.H., Li, X., Fei, D.Q., Zhao, C.Y., 2013. Molecular interaction of PCB153 to human serum albumin: insights from spectroscopic and molecular modeling studies. J. Hazard. Mater. 248, 313–321. Iwata, N., Mukai, T., Yamakoshi, Y.N., Hara, S., Yanase, T., Shoji, M., Endo, T., Miyata, N., 1998. Effects of C-60, a fullerene, on the activities of glutathione S-transferase and glutathione-related enzymes in rodent and human livers. Fuller. Sci. Technol. 6, 213–226. Johansson, H.K.L., Hansen, J.S., Elfving, B., Lund, S.P., Kyjovska, Z.O., Loft, S., Barfod, K.K., Jackson, P., Vogel, U., Hougaard, K.S., 2017. Airway exposure to multi-walled carbon nanotubes disrupts the female reproductive cycle without affecting pregnancy outcomes in mice. Part. Fibre Toxicol. 14, 17. Kamanga-Sollo, E., Thornton, K.J., White, M.E., Dayton, W.R., 2017. Role of G proteincoupled estrogen receptor-1 in estradiol 17β -induced alterations in protein synthesis and protein degradation rates in fused bovine satellite cell cultures 1. Domest. Anim. Endocrinol. 58, 90–96. Karelson, M., Martin, D., 2010. The quantitative structure activity relationships for predicting HIV protease inhibition by substituted fullerenes. Lett. Drug Des. Discov. 7, 587–595. Leal, M.P., Assali, M., Cid, J.J., Valdivia, V., Franco, J.M., Fernandez, I., Pozo, D., Khiar, N., 2015. Synthesis of 1D-glyconanomaterials by a hybrid noncovalent-covalent functionalization of single wall carbon nanotubes: a study of their selective interactions with lectins and with live cells. Nanoscale 7, 19259–19272. Lu, L.P., Zhan, T.J., Ma, M., Xu, C., Wang, J.P., Zhang, C.L., Liu, W.P., Zhuang, S.L., 2018. Thyroid disruption by bisphenol S analogues via thyroid hormoner receptor β: in vitro, in vivo and molecular dynamics simulation study. Environ. Sci. Technol. 52, 6617–6625. Matsumoto, N., Chen, G.H., Yumura, M., Futaba, D.N., Hata, K., 2015. Quantitative assessment of the effect of purity on the properties of single wall carbon nanotubes. Nanoscale 7, 5126–5133. Oner, D., Ghosh, M., Bove, H., Moisse, M., Boeckx, B., Duca, R.C., Poels, K., Luyts, K., Putzeys, E., Van Landuydt, K., Vanoirbeek, J.A.J., Ameloot, M., Lambrechts, D., Godderis, L., Hoet, P.H.M., 2018. Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition. Part. Fibre Toxicol. 15, 11. Pestana, D., Teixeira, D., Faria, A., Domingues, V., Monteiro, R., Calhau, C., 2015. Effects of environmental organochlorine pesticides on human breast cancer: putative involvement on invasive cell ability. Environ. Toxicol. 30, 168–176. Qu, J.H., Lu, C.C., Xu, C., Chen, G., Qiu, L.L., Jiang, J.K., Ben, S., Wang, Y.B., Gu, A.H., Wang, X.R., 2016. Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice. Environ. Toxicol. Pharmacol. 45, 150–157. Rancan, F., Gao, Q., Graf, C., Troppens, S., Hadam, S., Hackbarth, S., Kembuan, C., Blume-Peytavi, U., Ruhl, E., Lademann, J., Vogt, A., 2012. Skin penetration and cellular uptake of amorphous silica nanoparticles with variable size, surface functionalization, and colloidal stability. Acs Nano 6, 6829–6842. Ray, A., Macwan, I., Singh, S., Silwal, S., Patra, P., 2018. A computational approach for understanding the interactions between graphene oxide and nucleoside diphosphate kinase with implications for heart failure. Nanomaterials 8, 57. Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738. Ryoo, S.R., Kim, Y.K., Kim, M.H., Min, D.H., 2010. Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. Acs Nano 4, 6587. Shulin, Z., Chunlong, Z., Weiping, L., 2014. Atomic insights into distinct hormonal activities of Bisphenol A analogues toward PPARγ and ERα receptors. Chem. Res. Toxicol. 27, 1769. Suh, W.H., Suslick, K.S., Stucky, G.D., Suh, Y.H., 2009. Nanotechnology, nanotoxicology, and neuroscience. Prog. Neurobiol. 87, 133–170. Wang, D.Q., Meng, L.J., Fei, Z.F., Hou, C., Long, J.G., Zeng, L., Dyson, P.J., Huang, P.,
Fig. 7. Serum estradiol levels in blood from rats treated as control, E2 exposed and SWCNT exposed groups. Data are presented as mean ± S.E. of 8 rat in each group. * indicates significant difference when the values were compared to that of control group (p < 0.05). ** represents the significance of P < 0.01 when the values were compared to that of control group.
which suggested a potential estrogen interference effect of SWCNT. 4. Conclusion In this paper, fluorescence quenching experiments, molecular dynamics simulations and in vivo analysis were used to explore the interaction between SWCNTs and rat ERα. Fluorescence quenching experiments and molecular dynamic modeling demonstrated that SWCNT could bind to ERα and thus led to a conformational change on ERα tertiary structure levels. A further in vivo process presented that the exposure of SWCNT increased ERα expression and suggested a potential estrogen interference effect of SWCNT. In depth of insight into binding details between ERα and SWCNT was a necessary step to understand and identify recognition mechanism between SWCNT and proteins, thus be helpful to accurately evaluate the potential health risks of SWCNT. Acknowledgements This work was financially supported by the Research Center for EcoEnvironmental Sciences, Chinese Academy of Sciences, China (KF201517); Key Laboratory of Chemistry and Quality for Traditional Chinese Medicines of the University of Gansu Province, Gansu University of Chinese Medicines of China (zzy-2016-01); Gansu Provincial Administration of traditional Chinese Medicine, China (GZK-201763)，Lanzhou talent innovation and entrepreneurship technology program, China (2016-RC-19). Conflicts of interest There are no potential conflicts of interest to disclose. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2019.01.101. References Acaz-Fonseca, E., Sanchez-Gonzalez, R., Azcoitia, I., Arevalo, M.A., Garcia-Segura, L.M., 2014. Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators. Mol. Cell. Endocrinol. 389, 48–57. Beddoes, C.M., Case, C.P., Briscoe, W.H., 2015. Understanding nanoparticle cellular entry: a physicochemical perspective. Adv. Colloid Interface Sci. 218, 48–68. Bisesi, J.H., Robinson, S.E., Lavelle, C.M., Ngo, T., Castillo, B., Crosby, H., Liu, K.R., Das, D., Plazas-Tuttle, J., Saleh, N.B., Ferguson, P.L., Denslow, N.D., Sabo-Attwood, T., 2017. Influence of the gastrointestinal environment on the bioavailability of ethinyl estradiol sorbed to single-walled carbon nanotubes. Environ. Sci. Technol. 51, 948–957. Campagnolo, L., Massimiani, M., Palmieri, G., Bernardini, R., Sacchetti, C., Bergamaschi, A., Vecchione, L., Magrini, A., Bottini, M., Pietroiusti, A., 2013. Biodistribution and
378
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
chlorothioxanthone and its toxic effects on mRNA and protein expression and activities of human CYP1A2 and CYP3A4 enzymes. Environ. Sci. Technol. 52, 11904–11912. Zhang, J.Y., Liu, R., Niu, L.L., Zhu, S.Y., Zhang, Q., Zhao, M.R., Liu, W.P., Liu, J., 2018. Determination of endocrine-disrupting potencies of agricultural soils in China via a battery of steroid receptor bioassays. Environ. Pollut. 234, 846–854. Zhang, L.Y., Ren, X.M., Wan, B., Guo, L.H., 2014. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor gamma. Toxicol. Appl. Pharmacol. 279, 275–283. Zhang, Y.B., Ali, S.F., Dervishi, E., Xu, Y., Li, Z.R., Casciano, D., Biris, A.S., 2010. Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. Acs Nano 4, 3181–3186. Zhu, Z.W., Schuster, D.I., Tuckerman, M.E., 2003. Molecular dynamics study of the connection between flap closing and binding of fullerene-based inhibitors of the HIV1 protease. Biochemistry 42, 1326–1333. Zhuang, S.L., Wang, H.F., Ding, K.K., Wang, J.Y., Pan, L.M., Lu, Y.L., Liu, Q.J., Zhang, C.L., 2016. Interactions of benzotriazole UV stabilizers with human serum albumin: atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations. Chemosphere 144, 1050–1059.
2018. Multi-layered tumor-targeting photothermal-doxorubicin releasing nanotubes eradicate tumors in vivo with negligible systemic toxicity. Nanoscale 10, 8536–8546. Wang, W.W., Jiang, C.J., Zhu, L.D., Liang, N.N., Liu, X.J., Jia, J.B., Zhang, C.K., Zhai, S.M., Zhang, B., 2014. Adsorption of bisphenol A to a carbon nanotube reduced its endocrine disrupting effect in mice male offspring. Int. J. Mol. Sci. 15, 15981–15993. Warnmark, A., Treuter, E., Gustafsson, J.A., Hubbard, R.E., Brzozowski, A.M., Pike, A.C.W., 2002. Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha. J. Biol. Chem. 277, 21862–21868. Xu, Y., Luo, Z., Li, S.X., Li, W.G., Zhang, X.R., Zuo, Y.Y., Huang, F., Yue, T.T., 2017. Perturbation of the pulmonary surfactant monolayer by single-walled carbon nanotubes: a molecular dynamics study. Nanoscale 9, 10193–10204. Yao, P.L., Ehresman, D.J., Rae, J.M.C., Chang, S.C., Frame, S.R., Butenhoff, J.L., Kennedy, G.L., Peters, J.M., 2014. Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid. Toxicology 326, 62–73. Yue, T.T., Zhang, X.R., 2012. Cooperative effect in receptor-mediated endocytosis of multiple nanoparticles. Acs Nano 6, 3196–3205. Zhan, T.J., Pan, L.M., Liu, Z.F., Chen, J.Y., Ge, Z.W., Lu, L.P., Zhang, X.F., Cui, S.X., Zhang, C.L., Liu, W.P., Zhuang, S.L., 2018. Metabolic susceptibility of 2-
379
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Understanding the interaction of single-walled carbon nanotube (SWCNT) on estrogen receptor: A combined molecular dynamics and experimental study
T
Xinhe Liua, Tingting Liub, Juanjuan Songc, Ying Haia, Feng Luand, Haixia Zhange, Yongna Yuanf, ⁎ Hongyu Lia, Chunyan Zhaoa, a
School of Pharmacy, Lanzhou University, Lanzhou 730000, China Gansu Provincial Maternity and Child-care Hospital, Lanzhou 730000, China Pulmonary Hospital of Lanzhou, Lanzhou 730000, China d College of Chemistry and Chemical Engineering, Yantai University, Yantai 264000, China e College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China f School of Information Science & Engineering, Lanzhou University, Lanzhou, 730000, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Single-walled carbon nanotube (SWCNT) Estrogen receptor (ER) Molecular dynamics Fluorescence
Considering the large-scale production of diversified nanomaterials, it is paramount importance to unravel the structural details of interactions between nanoparticles and biological systems, and thus to explore the potential adverse impacts of nanoparticles. Estrogen receptors (ER) is one of the most important receptor of human reproductive system and the binding of carbon nanotubes to estrogen receptors was the possible trigger leading to the reproductive toxicity of carbon nanotubes. Thus, with single-walled carbon nanotube (SWCNT) treated as model nanomaterials, a combination of in vivo experiments, spectroscopy assay and molecular dynamic modeling was applied to help us unravel some important issues on the binding characterization between SWCNT and the ligand binding domain (LBD) of ER alpha (ERα). The fluorescence assay and molecular dynamics simulations together validated the binding of SWCNT to ERα, suggesting the possible molecular initiating event. As a consequence, SWCNT binding led to a conformational change on tertiary structure levels and hydrophobic interaction was recognized as the driving force governing the binding behavior between SWCNT and LBD of ERα. A in vivo process presented that the exposure of SWCNT increased ERα expression from 26.43 pg/ml to 259.01 pg/ml, suggesting a potential estrogen interference effects of SWCNT. Our study offers insight on the binding of SWCNT and ERα LBD at atomic level, helpful to accurately evaluate the potential health risks of SWCNT.
1. Introduction The number of nanomaterials products has been keeping increasing over the past decade (Guo et al., 2017; Wang et al., 2018). Nanoparticles also have great potential in biomedical applications such as drug delivery, radiation therapy, chemotherapy and biosensors (Gaunt et al., 2015; Matsumoto et al., 2015; Ray et al., 2018). However, the biological effects of nanomaterials are extremely unpredictable (Suh et al., 2009). In particular, it can interact with biological macromolecules. Therefore, proteins have always been a research focus in the interaction between nanomaterials and biomacromolecules (Chang et al., 2017; Leal et al., 2015; Yue and Zhang, 2012). For example, it is reported that fullerene (C60) and single-walled carbon nanotubes
⁎
(SWCNT) can effectively interacting with the hydrophobic binding sites on HIV-1 protease (HIV-1P)] and glutathione-S-transferase (GST), which leads to a suppression the activity of enzymes (Iwata et al., 1998; Karelson and Martin, 2010; Zhu et al., 2003). On the other sides, an injected nanoparticle into a living system could also result in an uncountable number of interactions (Beddoes et al., 2015; Oner et al., 2018). The highly unintended or undesired interactions of nanomaterials with biological molecules will cause side effects with a strong possibility, which thus may induce so-called cytotoxicity. Now, more and more nanotoxicology researches have been focused on the understanding the details of unintended or undesired interactions of nanomaterials with biological molecule (Chen et al., 2015; Chortarea et al., 2017; Rancan et al., 2012). Growing number of studies have proved
Corresponding author. E-mail address: [email protected] (C. Zhao).
https://doi.org/10.1016/j.ecoenv.2019.01.101 Received 30 October 2018; Received in revised form 21 January 2019; Accepted 29 January 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
2.2. Fluorescence measurement
that NPs can cause adverse physiological effects such as inflammation and immunological responses that lead to dysfunction of the tissues and organs. For example, Zuo et al. summarized the interactions between proteins and carbon nanotubes (CNTs), which should be a necessary step to understand the molecular mechanisms of nanotoxicity. At present, as the most commonly used nanomaterial, carbon nanotubes have recently been reported to have reproductive toxicity (Xu et al., 2017; Zhang et al., 2010). It has been reported that intravenous injection of carbon nanotubes into adult female mice causes transient histopathological changes (Wang et al., 2014). Intraperitoneal injection of multiwalled nanotubes in mice can cause malformations, miscarriage and can also increase the serum estradiol levels (Campagnolo et al., 2013; Ryoo et al., 2010). Estrogen receptors is one of the important parts of human reproductive system, the interaction between carbon nanotubes and estrogen receptors may be one of the important reasons for the reproductive toxicity of carbon nanotubes (Bisesi et al., 2017; Johansson et al., 2017). As ligand-regulated transcription factors, the biological effects of estrogen receptors are mediated primarily by binding to the endogenous estrogen estradiol-17β (E2) (Acaz-Fonseca et al., 2014; Kamanga-Sollo et al., 2017). However, many structurally diverse exogenous compounds, including phytochemicals, hydroxylated polychlorinated biphenyls, pesticides and other industrial compounds have been reported to bind to estrogen receptors and prevent unexpected estrogen-like activities (Collins-Burow et al., 2012; Pestana et al., 2015; Zhang et al., 2018). It may be one of the reasons that affects the reproductive system and leads to reproductive toxicity. For carbon nanotubes, given that it is structurally compatible with estrogen receptors, we hypothesized that, similar to other exogenous chemicals, carbon nanotubes can also interact with estrogen receptors to activate estrogen receptors and affect the reproductive system. However, the details of the interaction between carbon nanotubes and estrogen receptors are still unclear. In fact, an understanding of the mechanisms of estrogen receptor and carbon nanotubes binding may be a key stage in exploring how reproductive toxicity occurs and predicting the potential for adverse biological effects. Therefore, in this study, three methods were applied, including molecular dynamics simulations, fluorescence experiments and animal experiments to reveal the structural properties and details of the interaction between single-walled nanotubes and estrogen receptors. Given that evaluating the potential adverse biological effects of nanomaterials is much more complex, computer-based methods have been constructed that can help us understand the mechanisms of action of toxicity and the molecular details of their interactions with biomolecules. Due to the wide application of carbon nanotube materials, it is particularly important to study the structural characteristics and details of the interaction between nanomaterials and biological systems at the molecular level, thus further predicting and evaluating the potential toxicological effects of SWCNTs.
The instrument used in the fluorescence titration experiment was Model RF-5301 (Shimadzu Corporation, Japan). The fluorescence excitation wavelength was set to 385 nm and the slit width was set to λem = 5 nm and λex = 5 nm. Coumestrol (CS) was selected as the fluorescence spectroscopy probe targeting estrogen receptor. Concentration of ERα solution was fixed at 0.8 μM (2.64 × 10−2 g/L) with PBS buffer of 0.1 M and PH = 7. A targeted estrogen receptor fluorescent probe was added at a concentration of 0.1 μM (2.68 × 10−5 g/L) CS. The SWCNT suspension was gradually titrated to a final concentration of 0, 10−4 g/L, 2 × 10−4 g/L, 4 × 10−4 g/L, 6 × 10−4 g/L, 8 × 10−4 g/L. Fluorescence intensity was then recorded at a fluorescence excitation wavelength of 385 nm with a time interval of ten minutes (Han et al., 2013; Zhang et al., 2014; Zhuang et al., 2016). 2.3. Molecular dynamics The protein sequence of Rattus norvegicus ERα (rERα) was retrieved from the NCBI (http://www.ncbi.nlm.nih.gov) with sequence number of BAI48013. Homology models of rERα ligand-binding domain were constructed using Swiss-model with human ERα (hER PDB ID: 1GWR) as the template. More information about homology modeling presented in Roy et al. (2010). The identified best model was based on highest C-score, which was obtained from the relative clustering structural density and consensus significance. Besides it, Ramachandran plots calculated by Rampage program were used to explore the stereo chemical quality of the constructed ERα models. The initial 3D structures of rERα obtained from homology modelings were refined by 1 ns MD simulation with FF03 force field. The structure of the single-walled nanotubes was constructed using the Nanotube Builder module in the VMD software. The chirality m = n = 5 and the length was set to 1.0 nm. The constructed single-walled nanotubes contained 100 carbon atoms in total. Docking of singlewalled nanotubes to constructed rERα was performed using Autodock software 4.2.5.1. Molecular dynamics simulation of 50 ns was then applied to the SWCNT-ERα system using Amber14, in which the ERα protein was processed using FF03 force field and SWCNT was processed using GAFF force field. The module Tleap was used to hydrogenate the composite SWCNT-ERα system, which was then dissolved into a TIP3P periodic regular hexahedral water box to set any atom in the composite SWCNT-ERα. The minimum distance of the water box boundary is a minimum of 5 Å. The water molecules in the solution are replaced with positively charged sodium ions to make the entire system electrically neutral. All key lengths are limited by the SHAKE algorithm. The Verlet Frog Leaping Algorithm is used to set the integration step in the whole process of the dynamics to 2 fs. The limiting force for setting the composite SWCNT-ERα was set to 100 kcal/mol Å2 for water. The optimization of molecules and some counter particles was first performed using the steepest descent method of 3000 steps followed by a 2000 steps conjugate gradient method. The whole system was then optimized using the first method without any restriction. Subsequently, the temperature of the entire system was increased from 0 K to 298 K within 50 ps. Then a 100 ns dynamic simulation was performed at a 298 K NPT system (Ding et al., 2017; Lu et al., 2018; Shulin et al., 2014). The average structures derived from the last 30 ns trajectory of ERαSWCNT system were extracted for the further analysis. The Ramachandran plot was performed by the Rampage program. The distance and contact area were calculated using the Chimera 1.13.1. The residue-residue interaction networks (RIN) analysis was performed by Cytoscape 3.4.0 and the plugin RINalyzer. The binding energy of SWCNT-ERα system was calculated by the method about MM-GBSA. The Root Mean Square Deviation (RMSD) and Root-mean-square fluctuation (RMSF) analyses were performed by Origin8. The interhelical angles of H12 was calculated using the g_angle procedure in GROMACS 5.0 software.
2. Materials and methods 2.1. Chemicals and test solutions The Single-walled nanotubes solutions (1 g/L) were purchased from Shenzhen Nangang Port. Co., Ltd. (Shenzhen, China). The estrogen receptor fluorescent probe Coumestrol (CS) was purchased from J&K Blingway Technology Co., Ltd. Elisa kit (Rat Estradiol, E2 ELISA Kit) was purchased from Wuhan Liuhe Biotechnology Co., Ltd. The estrogen recombinant proteins (200 μg/ml) were purchased from Shanghai Xinyu Co., Ltd., containing recombinant ERα ligand binding domain (LBD) protein ERα of amino acids 178–483 of rat ERα (rERα248–484) with two N-terminal tags. The SD (Sprague Dawley) rats, aged 2–3 weeks, were purchased from the Animal Experimental Center of Lanzhou University. 374
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
2.4. In vivo experimental procedures Twenty-four female SD rats with rat age 3–4 weeks and the weight at 180 ± 20 g rats were divided into three groups, including singlewalled nanotubes (SWCNT) group, estradiol (E2) group and blank control group. After one week of acclimatization, the eight rats of SWCNT group were intragastrically administered as a single-walled nanotube suspension at a concentration of 15 mg/kg/d. The eight rats of E2 group were intragastrically administered as estradiol solution at 200 μg/kg/d and the eight rats in blank control group were administered as normal saline. Fig. 1. The fluorescence of ERα (0.8 μM) and SWCNT (1–6) at concentrations of 0 (only ERα), 10−4 g/L, 2 × 10−4 g/L, 4 × 10−4 g/L, 6 × 10−4 g/L, 8 × 10−4 g/L. The scanning wavelength ranged from 300 nm to 500 nm, the excitation wavelength was 385 nm, and the measurement temperature was 298 K.
2.4.1. Serum estradiol levels measurement After four weeks of gavage, the eyeballs of the above 24 SD rats were bled and centrifuged. The serum was collected and the serum estradiol levels were tested using the Elisa kit (Rat Estradiol, E2 ELISA Kit) (Qu et al., 2016).
residues in favored regions, 1.3% residues in allowed regions, and 0.4% residues in outlier regions. Ramachandran plots proved that the constructed rERα model was reasonable. The binding site of hERα was defined as the residues around 9 Å distance of the ligand estradiol. Considering that the similarity of rERα and hERα proteins, the binding sites of rERα were characterized as the same regions. Docking of singlewalled nanotubes to constructed rERα was performed. Molecular dynamic (MD) simulations were further used to explore the biological processes between the SWCNT and the ERα. The stability of the entire complex system was analyzed by root-mean-square deviations (RMSD) analysis (Fig. S2). The RMSD was calculated by extracting the relevant data from the trajectory file. It can be seen that the ERα-SWCNT system has reached basic stability with no significant fluctuations after 40 ns. The average structures derived from the last 20 ns trajectory of ERα-SWCNT system were then extracted for the further analysis.
2.4.2. Western blot As shown, the SD rats were sacrificed by spinal dislocation. The uterine tissues were taken and stored in a cryogenic freezer at −70 °C. The tissue sample was added into cold Lysis buffer and centrifuged. The supernatant was taken as a whole protein extract and protein ration was tested using Bradford method. The extracted protein was then transferred to a nitrocellulose (NC) filter. Membranes were blocked with blocking buffer (5% nonfat dry milk in Tween-20 (TBST) Tris buffered saline) and incubated with TBST after 30 min of incubation at room temperature. The membranes were then incubated against primary antibody of ERα rabbit polyclonal antibody at 4 °C overnight. After overnight incubation, the membranes were washed three times at room temperature with TBST and then incubated with a biotinylated secondary antibody (rabbit anti-GAPDH (10B8)) for 2 h at room temperature. The secondary antibody was then washed with TBST and the color was imaged. The imaging was performed using G:BOX chemiXR5 software and gray-scale analysis using Gel-Pro32 software. The expression of glycoaldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control and the density ratio of protein density to GAPDH for grayscale analysis were calculated (Yao et al., 2014; Zhan et al., 2018). The statistical analysis of Estradiol (E2) level assay was applied using the SPSS 11.5 statistical software package, with value of p < 0.05 defined as significance. Parametric comparison was performed by one-way Analysis of Variance (ANOVA) followed by Dunnett-t-test.
3.2.2. Binding modes of SWCNT to ERα Molecular dynamics (MD) simulations were used to explore the model dynamic fluctuations in the structure of ERα upon binding to the single-wall carbon nanotube. This binding process between SWCNT and ERα can be illustrated by the interface area between SWCNT and the ER protein domain (Fig. 2A). The parameter of interface area S was defined as half of the difference between the solvent-accessible surface area of the complex and the sum of solvent-accessible surface areas of the protein-SWCNT complex. The important SWCNT-ERα complex snapshots at different times were displayed to present the process of SWCNT plugging into ER. At the beginning, the value of interface area S presented a very small value of 334 Å2. The area S increased very quickly to 340 Å2 within the first 5 ns, presenting that binding sites of ER and the SWCNT approached each other very quickly. The area S slowly increased from 340 to 345 Å2 in the next 10 ns. The area S increased reached its maximal value of 354 Å2 at 25 ns. After 35 ns, it is basically
3. Result and discussion 3.1. Fluorescence analysis for SWCNT-ERα LBD interaction Fluorescence titration experiment is an effective method to explore the effects of single-walled nanotubes (SWCNT) on the structural changes of LBD of ERα. As shown, fluorescence spectrum of the estrogen receptor was recorded in the presence of SWCNTs (Fig. 1). Fluorescent probe CS was added to increase the fluorescence intensity of the estrogen receptor and the fluorescence emission spectra were measured as the average of three scans at ten minute intervals. As presented, as the concentrations of SWCNT increasing, the fluorescence intensity of the ERα gradually decreased. The strong fluorescence quenching clearly showed the interaction between the SWCNT and the estrogen receptor. It also reflected that tertiary structures of ERα have changed due to the addition of SWCNTs.
Fig. 2. (A) The SWCNT-ERα interface areas as a function of time. Some representative snapshots (5 ns, 15 ns, 25 ns and 35 ns) were also presented. SWCNT was presented as gold and rERα was presented as blue. (B) Binding modes of SWCNT in the binding pocked of ERα. Structures of ERα were presented as ribbon. The blue ribbon represented free bound ERα protein and the golden ribbon represented SWCNT-ERα.
3.2. Molecular dynamics modeling 3.2.1. Homology modeling The constructed homology model of rERα was examined by the Ramachandran plot (Fig. S1). The rERα model presented 98.3% 375
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
were all located near the binding cavity. The above 15 amino acids can further be classified as polar amino acids and non-polar amino acids. Among the above 15 amino acids that contributed greatly, there were only 2 polar amino acids, but 13 non-polar amino acids, accounting for 86.7% of the total. As known, the polar amino acids have strong hydrophilicity, while the nonpolar polar amino acids have strong hydrophobicity. Therefore, it can be seen (Fig. 3), that the contribution of hydrophobic interaction was the main force of binding behavior, while electrostatic interaction contributed very little and almost no contribution. Based on these results, it can be concluded that hydrophobic interactions can be attributed to the driving forces that play an important role in ERα-SWCNT binding.
straight, indicating that the contact surface of the domain has reached a relatively stable state. It suggested that SWCNT was finally successfully inserted into the bonding cavity of rERα and remained stable.
3.2.3. Binding free energy and energy decomposition In order to judge the binding ability of single-walled nanotubes (SWCNT) to ERα, MMGBSA method was used to calculate the binding free energy of the ERα-SWCNT system, which presented as ΔGtotal = −34.51 kcal/mol in Table S1 (Supporting information). The total free energy can be decomposed into its components as shown in Table S1. The molecular mechanics electrostatic contribution (ΔEele ) was counterbalanced by the contribution of electrostatic of the solvation energy (ΔEgb ). A value of 7.21 kcal/mol showing that the total electrostatic contribution (ΔEgb + ΔEele ) was slightly non-favorable for the binding. The van derWaals contribution together with the non-polar solvation free energy (ΔE vdws + ΔEsurf ) constituted the non-polar energy term and a value of −41.72 kcal/mol presented a significant favorable contribution to the overall binding free energy. As known, the total binding free energy value was more negative and the combination of between SWCNT and ERα was better. Here, the relatively low value of the total energy suggested that the binding affinity between SWCNT and ERα was quite strong and the binding was stable. The result of the total energy analysis was also consistent with the fluorescence titration experiment, demonstrating a strong combination of SWCNT to the ligand binding domain of ERα. In addition, the negative value of ΔE vdws indicates that the hydrophobic interaction between SWCNT and ERα promoted favorable binding. The highly favorable non-polar component originated from the van derWaals energy, which also demonstrated hydrophobic interactions as a key force governing the bindings between SWCNT and ERα. Besides it, energy contribution difference analysis was performed to rationalize the relative importance of contributions for each residue (Fig. 3). It was found that there were 15 amino acids that contributed a lot, namely Ser214, His220, Leu42, Leu80, Leu83, Leu87, Leu124, Leu221, Met117, Met213, Met218, Ile120, Phe121 and Gly217, which
3.2.4. Residue interaction network The residue interaction network (RIN) is a new technology to identify key amino acid interactions and to identify the topological structure of the entire system (Fig. 4). RIN is a modern topology based analysis which helped in identification of residue–residue contact difference in biological systems, where the nodes represent amino acid residues and the edges represent the interactions between the residues. The structure of the ERα was extracted from the two system dynamics of SWCNT binding ERα (SWCNT- ERα) system (Fig. 4A) and the free bound ERα (Free-ERα) (Fig. 4B), respectively. As shown, in the free bound ERα, the total amino acid-amino acid interactions were 551, while in the SWCNT-ERα system, the total number of interactions was reduced to 497 with a reduction of 9.1%. The results presented that the binding of SWCNTs relaxed the structure of the protein. In order to further analyze the interactions between amino acids, we selected all the amino acids in the surrounding 5 Å range of the binding pocket, with a total of 28 amino acids, as shown in (Fig. 5). For the free bound ERα system, the total edges were 50, of which 20 corresponded to van derWaals contacts, 1 to ionic interactions and 29 to hydrogen bonds. While, in the SWCNT-ERα system, the total edges was reduced to 26, the hydrogen bonds was 5, the van derWaals force was 21 and the ionic interaction was 0, which showed the total decreasing rate of 48%, the hydrogen bonds decreasing rate of 75.0% and the van derWaals forces decreasing rate of 27.6%. In summary, the combination of SWCNTs severely damaged the mutual network of ERα residues, especially to hydrogen bonds, which resulted in the loosening of the overall ERα structure and the destruction of the secondary and tertiary structure of the protein. 3.2.5. H12 rotations analysis The root mean square fluctuations (RMSF) plot for the helix 12 residues and the surrounding residues was presented (Fig. S3). As seen, the residues in the region Helix 12 (228–236) showed higher fluctuations compared to the adjacent regions. One possible reason is that, H12, as the most important helix of ERα, its position had changed during the simulation, indicating SWCNT had already modified the conformation of H12. Thus, the movement and rotations of H12 were further explored because the position of H12 in the LBD of ERαs is the key factor governing the relative recruitment of coregulators to coordinate a transcriptional response (Warnmark et al., 2002). The structures of ERα complexed with agonist and antagonist ligands are induced into different conformations, particularly the position and orientation of H12 (Fig. 6A). The relative rotational orientations of H12 and interhelical angles were quantitatively examined in (Fig. 6B). As shown, the H12 in ERα with binding of SWCNT turned from free bound state to agonist state with an interhelical angle of 10.2°, which suggested that SWCNT presented an agonist effect in rat ERα. The analysis of the movement and rotational angle of Helix 12 indicated that SWCNT had agonist effect on rat ERα, which might lead to a further influence on the recruitment of coregulators and the subsequent transcriptional regulation.
Fig. 3. Binding energy analysis of SWCNT-ERα system. (A) Contribution of each amino acid to SWCNT and ERα. (B) Polarity (dark grey) and non-polar contribution (light gray) of key amino acids to binding freedom. Negative values are favorable for the combination of SWCNTs. 376
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
Fig. 4. Characterization of the residue-residue networks for the ERα residues of (A) Free bound ERα and (B) SWCNT-ERα system. SWCNT was presented using gold color. The red spheres presented residues of ERα. Lines indicated residue-residue interactions, including red lines present hydrogen bonds and blue lines present van derWaals force.
3.3. In vivo analysis 3.3.1. Western blot analysis To further analyze the effect of SWCNTs on the expression of estrogen receptor in rats, Western Blot immunoblot analysis was performed to determine the effect of SWCNTs on the expression of ERα in rat uterus. As can be seen (Fig. S4), the expression of ERα in the uterus of the rats administered E2 was slightly up-regulated compared with the control group, while the expression of ERα in the uterus of the rats treated with SWCNT alone was significantly down-regulated. This indicates that the addition of SWCNT significantly reduced the expression of ERα in the rat uterus. This also reflects the decrease in estrogen levels in the rat uterus after the addition of SWCNTs. This may cause changes in hormone levels in the uterus.
3.3.2. Estradiol (E2) level assay As known, estradiol (E2) level in serum is an important indicator to evaluate reproductive endocrine interference effect. To examine the effects of single-walled nanotubes on the reproductive system, estradiol in rat serum was measured. As shown in (Fig. 7), the level of estradiol in the blank control group was 26.43 pg/ml, followed by 259.01 pg/ml in the SWCNT group and 690.48 pg/ml in E2 group. The level of estradiol in the rat serum administered with the SWCNT group was much higher than that of the blank control group. It indicated that the addition of the SWCNTs raised the level of estradiol in the rats. Estradiol is an extremely important hormone in the body. Changes in estradiol levels in the body may be related to hormone feedback and pituitary regulation,
Fig. 6. (A) Positions of the H12 (blue ribbon) in free bound ERα system, agonist-bound and antagonist-bound state. The ER LBD was presented as red ribbon in free bound state and presented as electrostatic surface in agonist-bound and antagonist-bound state. (B) Interhelical angles of H12 of free bound ERα system and SWCNT-ERα complex. The interhelical angles were defined as the rotational angle of H12 from free bound state (blue color) to agonist state (golden color).
Fig. 5. Characterization of the residue-residue networks for the ERα residues surrounding 5 Å range of the binding pocket from (A) Free bound ERα; (B) SWCNT-ERα system. The yellow spheres presented residues of ERα. SWCNT was presented using gold color. Red lines indicate hydrogen bonds. Blue lines indicated van derWaals force and gray lines indicated ion interactions.
377
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
toxicity of pegylated single wall carbon nanotubes in pregnant mice. Part. Fibre Toxicol. 10, 21. Chang, L.F., He, X.W., Chen, L.X., Zhang, Y.K., 2017. A novel fluorescent turn-on biosensor based on QDs@GSH-GO fluorescence resonance energy transfer for sensitive glutathione S-transferase sensing and cellular imaging. Nanoscale 9, 3881–3888. Chen, S.M., Xiong, C.Q., Liu, H.H., Wan, Q.Q., Hou, J., He, Q., Badu-Tawiah, A., Nie, Z.X., 2015. Mass spectrometry imaging reveals the sub-organ distribution of carbon nanomaterials. Nat. Nanotechnol. 10, 176–182. Chortarea, S., Barosova, H., Clift, M.J.D., Wick, P., Petri-Fink, A., Rothen-Rutishauser, B., 2017. Human asthmatic bronchial cells are more susceptible to subchronic repeated exposures of aerosolized carbon nanotubes at occupationally relevant doses than healthy cells. Acs Nano 11, 7615–7625. Collins-Burow, B.M., Antoon, J.W., Frigo, D.E., Elliott, S., Weldon, C.B., Boue, S.M., Beckman, B.S., Curiel, T.J., Alam, J., McLachlan, J.A., Burow, M.E., 2012. Antiestrogenic activity of flavonoid phytochemicals mediated via the c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha. J. Steroid Biochem. Mol. Biol. 132, 186–193. Ding, K.K., Kong, X.T., Wang, J.P., Lu, L.P., Zhou, W.F., Zhan, T.J., Zhang, C.L., Zhuang, S.L., 2017. Side chains of parabens modulate antiandrogenic activity: in vitro and molecular docking studies. Environ. Sci. Technol. 51, 6452–6460. Gaunt, N.P., Patil-Sen, Y., Baker, M.J., Kulkarni, C.V., 2015. Carbon nanotubes for stabilization of nanostructured lipid particles. Nanoscale 7, 1090–1095. Guo, Q., You, H.H., Yang, X., Lin, B.C., Zhu, Z.H., Lu, Z.S., Li, X.X., Zhao, Y., Mao, L., Shen, S.P., Cheng, H.Y., Zhang, J.T., Deng, L.F., Fan, J.H., Xi, Z.G., Li, R., Li, C.M., 2017. Functional single-walled carbon nanotubes 'CAR' for targeting dopamine delivery into the brain of parkinsonian mice. Nanoscale 9, 10832–10845. Han, C., Fang, S.B., Cao, H.M., Lu, Y., Ma, Y.Q., Wei, D.F., Xie, X.Y., Liu, X.H., Li, X., Fei, D.Q., Zhao, C.Y., 2013. Molecular interaction of PCB153 to human serum albumin: insights from spectroscopic and molecular modeling studies. J. Hazard. Mater. 248, 313–321. Iwata, N., Mukai, T., Yamakoshi, Y.N., Hara, S., Yanase, T., Shoji, M., Endo, T., Miyata, N., 1998. Effects of C-60, a fullerene, on the activities of glutathione S-transferase and glutathione-related enzymes in rodent and human livers. Fuller. Sci. Technol. 6, 213–226. Johansson, H.K.L., Hansen, J.S., Elfving, B., Lund, S.P., Kyjovska, Z.O., Loft, S., Barfod, K.K., Jackson, P., Vogel, U., Hougaard, K.S., 2017. Airway exposure to multi-walled carbon nanotubes disrupts the female reproductive cycle without affecting pregnancy outcomes in mice. Part. Fibre Toxicol. 14, 17. Kamanga-Sollo, E., Thornton, K.J., White, M.E., Dayton, W.R., 2017. Role of G proteincoupled estrogen receptor-1 in estradiol 17β -induced alterations in protein synthesis and protein degradation rates in fused bovine satellite cell cultures 1. Domest. Anim. Endocrinol. 58, 90–96. Karelson, M., Martin, D., 2010. The quantitative structure activity relationships for predicting HIV protease inhibition by substituted fullerenes. Lett. Drug Des. Discov. 7, 587–595. Leal, M.P., Assali, M., Cid, J.J., Valdivia, V., Franco, J.M., Fernandez, I., Pozo, D., Khiar, N., 2015. Synthesis of 1D-glyconanomaterials by a hybrid noncovalent-covalent functionalization of single wall carbon nanotubes: a study of their selective interactions with lectins and with live cells. Nanoscale 7, 19259–19272. Lu, L.P., Zhan, T.J., Ma, M., Xu, C., Wang, J.P., Zhang, C.L., Liu, W.P., Zhuang, S.L., 2018. Thyroid disruption by bisphenol S analogues via thyroid hormoner receptor β: in vitro, in vivo and molecular dynamics simulation study. Environ. Sci. Technol. 52, 6617–6625. Matsumoto, N., Chen, G.H., Yumura, M., Futaba, D.N., Hata, K., 2015. Quantitative assessment of the effect of purity on the properties of single wall carbon nanotubes. Nanoscale 7, 5126–5133. Oner, D., Ghosh, M., Bove, H., Moisse, M., Boeckx, B., Duca, R.C., Poels, K., Luyts, K., Putzeys, E., Van Landuydt, K., Vanoirbeek, J.A.J., Ameloot, M., Lambrechts, D., Godderis, L., Hoet, P.H.M., 2018. Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition. Part. Fibre Toxicol. 15, 11. Pestana, D., Teixeira, D., Faria, A., Domingues, V., Monteiro, R., Calhau, C., 2015. Effects of environmental organochlorine pesticides on human breast cancer: putative involvement on invasive cell ability. Environ. Toxicol. 30, 168–176. Qu, J.H., Lu, C.C., Xu, C., Chen, G., Qiu, L.L., Jiang, J.K., Ben, S., Wang, Y.B., Gu, A.H., Wang, X.R., 2016. Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice. Environ. Toxicol. Pharmacol. 45, 150–157. Rancan, F., Gao, Q., Graf, C., Troppens, S., Hadam, S., Hackbarth, S., Kembuan, C., Blume-Peytavi, U., Ruhl, E., Lademann, J., Vogt, A., 2012. Skin penetration and cellular uptake of amorphous silica nanoparticles with variable size, surface functionalization, and colloidal stability. Acs Nano 6, 6829–6842. Ray, A., Macwan, I., Singh, S., Silwal, S., Patra, P., 2018. A computational approach for understanding the interactions between graphene oxide and nucleoside diphosphate kinase with implications for heart failure. Nanomaterials 8, 57. Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738. Ryoo, S.R., Kim, Y.K., Kim, M.H., Min, D.H., 2010. Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. Acs Nano 4, 6587. Shulin, Z., Chunlong, Z., Weiping, L., 2014. Atomic insights into distinct hormonal activities of Bisphenol A analogues toward PPARγ and ERα receptors. Chem. Res. Toxicol. 27, 1769. Suh, W.H., Suslick, K.S., Stucky, G.D., Suh, Y.H., 2009. Nanotechnology, nanotoxicology, and neuroscience. Prog. Neurobiol. 87, 133–170. Wang, D.Q., Meng, L.J., Fei, Z.F., Hou, C., Long, J.G., Zeng, L., Dyson, P.J., Huang, P.,
Fig. 7. Serum estradiol levels in blood from rats treated as control, E2 exposed and SWCNT exposed groups. Data are presented as mean ± S.E. of 8 rat in each group. * indicates significant difference when the values were compared to that of control group (p < 0.05). ** represents the significance of P < 0.01 when the values were compared to that of control group.
which suggested a potential estrogen interference effect of SWCNT. 4. Conclusion In this paper, fluorescence quenching experiments, molecular dynamics simulations and in vivo analysis were used to explore the interaction between SWCNTs and rat ERα. Fluorescence quenching experiments and molecular dynamic modeling demonstrated that SWCNT could bind to ERα and thus led to a conformational change on ERα tertiary structure levels. A further in vivo process presented that the exposure of SWCNT increased ERα expression and suggested a potential estrogen interference effect of SWCNT. In depth of insight into binding details between ERα and SWCNT was a necessary step to understand and identify recognition mechanism between SWCNT and proteins, thus be helpful to accurately evaluate the potential health risks of SWCNT. Acknowledgements This work was financially supported by the Research Center for EcoEnvironmental Sciences, Chinese Academy of Sciences, China (KF201517); Key Laboratory of Chemistry and Quality for Traditional Chinese Medicines of the University of Gansu Province, Gansu University of Chinese Medicines of China (zzy-2016-01); Gansu Provincial Administration of traditional Chinese Medicine, China (GZK-201763)，Lanzhou talent innovation and entrepreneurship technology program, China (2016-RC-19). Conflicts of interest There are no potential conflicts of interest to disclose. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2019.01.101. References Acaz-Fonseca, E., Sanchez-Gonzalez, R., Azcoitia, I., Arevalo, M.A., Garcia-Segura, L.M., 2014. Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators. Mol. Cell. Endocrinol. 389, 48–57. Beddoes, C.M., Case, C.P., Briscoe, W.H., 2015. Understanding nanoparticle cellular entry: a physicochemical perspective. Adv. Colloid Interface Sci. 218, 48–68. Bisesi, J.H., Robinson, S.E., Lavelle, C.M., Ngo, T., Castillo, B., Crosby, H., Liu, K.R., Das, D., Plazas-Tuttle, J., Saleh, N.B., Ferguson, P.L., Denslow, N.D., Sabo-Attwood, T., 2017. Influence of the gastrointestinal environment on the bioavailability of ethinyl estradiol sorbed to single-walled carbon nanotubes. Environ. Sci. Technol. 51, 948–957. Campagnolo, L., Massimiani, M., Palmieri, G., Bernardini, R., Sacchetti, C., Bergamaschi, A., Vecchione, L., Magrini, A., Bottini, M., Pietroiusti, A., 2013. Biodistribution and
378
Ecotoxicology and Environmental Safety 172 (2019) 373–379
X. Liu et al.
chlorothioxanthone and its toxic effects on mRNA and protein expression and activities of human CYP1A2 and CYP3A4 enzymes. Environ. Sci. Technol. 52, 11904–11912. Zhang, J.Y., Liu, R., Niu, L.L., Zhu, S.Y., Zhang, Q., Zhao, M.R., Liu, W.P., Liu, J., 2018. Determination of endocrine-disrupting potencies of agricultural soils in China via a battery of steroid receptor bioassays. Environ. Pollut. 234, 846–854. Zhang, L.Y., Ren, X.M., Wan, B., Guo, L.H., 2014. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor gamma. Toxicol. Appl. Pharmacol. 279, 275–283. Zhang, Y.B., Ali, S.F., Dervishi, E., Xu, Y., Li, Z.R., Casciano, D., Biris, A.S., 2010. Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. Acs Nano 4, 3181–3186. Zhu, Z.W., Schuster, D.I., Tuckerman, M.E., 2003. Molecular dynamics study of the connection between flap closing and binding of fullerene-based inhibitors of the HIV1 protease. Biochemistry 42, 1326–1333. Zhuang, S.L., Wang, H.F., Ding, K.K., Wang, J.Y., Pan, L.M., Lu, Y.L., Liu, Q.J., Zhang, C.L., 2016. Interactions of benzotriazole UV stabilizers with human serum albumin: atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations. Chemosphere 144, 1050–1059.
2018. Multi-layered tumor-targeting photothermal-doxorubicin releasing nanotubes eradicate tumors in vivo with negligible systemic toxicity. Nanoscale 10, 8536–8546. Wang, W.W., Jiang, C.J., Zhu, L.D., Liang, N.N., Liu, X.J., Jia, J.B., Zhang, C.K., Zhai, S.M., Zhang, B., 2014. Adsorption of bisphenol A to a carbon nanotube reduced its endocrine disrupting effect in mice male offspring. Int. J. Mol. Sci. 15, 15981–15993. Warnmark, A., Treuter, E., Gustafsson, J.A., Hubbard, R.E., Brzozowski, A.M., Pike, A.C.W., 2002. Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha. J. Biol. Chem. 277, 21862–21868. Xu, Y., Luo, Z., Li, S.X., Li, W.G., Zhang, X.R., Zuo, Y.Y., Huang, F., Yue, T.T., 2017. Perturbation of the pulmonary surfactant monolayer by single-walled carbon nanotubes: a molecular dynamics study. Nanoscale 9, 10193–10204. Yao, P.L., Ehresman, D.J., Rae, J.M.C., Chang, S.C., Frame, S.R., Butenhoff, J.L., Kennedy, G.L., Peters, J.M., 2014. Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid. Toxicology 326, 62–73. Yue, T.T., Zhang, X.R., 2012. Cooperative effect in receptor-mediated endocytosis of multiple nanoparticles. Acs Nano 6, 3196–3205. Zhan, T.J., Pan, L.M., Liu, Z.F., Chen, J.Y., Ge, Z.W., Lu, L.P., Zhang, X.F., Cui, S.X., Zhang, C.L., Liu, W.P., Zhuang, S.L., 2018. Metabolic susceptibility of 2-
379