2019 Volume 44 Issue 10 Pages 711-720
Recent studies have demonstrated a relationship between the disruption of zinc homeostasis and the onset of diseases. However, little is known about the factors that disrupt zinc homeostasis. Here, we investigated the effects of β-naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor (AHR), on intracellular zinc levels. Human hepatoma HepG2 cells were treated with β-naphthoflavone for 3 days, and intracellular labile and total zinc levels were assessed through flow cytometry and inductively coupled plasma atom emission spectroscopy, respectively. The mRNA levels of zinc transporters were determined by real-time PCR. Treatment of cells with β-naphthoflavone induced a decrease in intracellular labile zinc in a dose-dependent manner, with significantly decreased levels observed at 1 µM compared with controls. Additionally, intracellular total zinc levels demonstrated a decreasing trend with 10 µM β-naphthoflavone. Zinc pyrithione recovered the decrease in intracellular labile zinc levels induced by β-naphthoflavone, while zinc sulfate had no effect. Moreover, significant decreases in the mRNA levels of zinc transporters ZnT10 and ZIP5 were observed in response to 10 µM β-naphthoflavone. These results demonstrated that β-naphthoflavone has the potential to disrupt zinc homeostasis in hepatocytes. Although the underlying mechanism remains to be determined, suppression of zinc transporter transcription through AHR activation may be involved in the β-naphthoflavone-induced disruption of intracellular zinc levels.
In humans, zinc is the second most abundant trace element after iron and plays an important role in cell growth, differentiation, apoptosis, and metabolism (Grüngreiff et al., 2016; Joazeiro and Weissman, 2000; Kadrmas and Beckerle, 2004; Pabo et al., 2001; Prasad, 1995; Vallee and Falchuk, 1993). More than 300 proteins contain zinc-interacting domains, and these domains are important for regulating various cellular functions. In the central nervous system, zinc ions are enriched in the synaptic vesicles of glutamatergic neurons and are co-released with glutamate into the synaptic cleft upon stimulation (Frederickson et al., 2000). Therefore, zinc ions are thought to function as neurotransmitters or modulators of neurotransmission (Bitanihirwe and Cunningham, 2009; Nakashima and Dyck, 2009; Paoletti et al., 2009). To perform these important biological roles, the intracellular zinc level is maintained within a narrow range in mammalian cells (Palmiter and Findley, 1995). Zinc homeostasis is precisely regulated by the balanced actions of zinc-binding proteins, metallothioneins, and zinc transporters (Hara et al., 2017; Hojyo and Fukada, 2016). Given the critical functions of zinc, perturbation of zinc levels would thus be detrimental to cell survival, and several studies have revealed alterations of serum, tissue, and cellular zinc concentrations during the onset of certain diseases, including type 2 diabetes (Yary et al., 2016), Alzheimer’s disease (Li and Wang, 2016; Portbury and Adlard, 2015), senile dementia (Kawahara et al., 2014), and cancer (Kolenko et al., 2013; Ressnerova et al., 2016). These findings suggest that zinc homeostasis plays an important role in the onset and progression of disease. However, little information is available regarding the factors that disrupt zinc homeostasis. Clarification of the factors and mechanisms that disrupt zinc homeostasis may thus provide important information for the prevention of disease and the development of novel therapies.
The aryl hydrocarbon receptor (AHR) belongs to a family of ligand-dependent basic helix-loop-helix transcription factors. Although exogenous ligand for the AHR has been identified, the biological functions of this receptor are not fully understood because the endogenous ligand for the AHR has not been identified. Numerous researches regarding the exploration of endogenous AHR ligand has been carried out, and various compounds, for example, tryptophan derivatives and other indole-containing compounds, such as kynurenine (Seok et al., 2018), 6-formylin-dolo[3,2-b]carbazole (Rannug et al., 1987; Wei et al., 1998) and 2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (Song et al., 2002), indirubin and indigo (Adachi et al., 2001), bilirubin (Sinal and Bend, 1997), 7-ketocholesterol (Savouret et al., 2001), and lipoxin A4 (Schaldach et al., 1999), have been reported as candidates. However, because of various issues, such as the low levels, lack of potency and restricted distribution, it remains unknown whether these compounds are a major endogenous regulator of AhR signaling. On the other hand, numerous studies, including analyses of AHR-null mice, have suggested that this receptor plays a role in the development of embryo, liver, and immune functions as well as in cell growth and differentiation (Fernandez-Salguero et al., 1996; Harrill et al., 2013; Kolluri et al., 1999; Mimura et al., 1997; Schmidt et al., 1996). Previous studies have demonstrated that 3,3’,4,4’,5-pentachlorobiphenyl (IUPAC No. PCB126), an exogenous ligand of AHR, suppresses alcohol dehydrogenase (Ishii et al., 2001), carbonic anhydrase III (Ikeda et al., 2000), and aldolase B (Ishii et al., 1997) in rat livers. All these proteins bind zinc (Arslanian et al., 1971; Berry and Marshall, 1993; Tupper et al., 1952; Vallee and Hoch, 1955), and zinc deficiency leads to decreased levels and activities of these proteins (Huber and Gershoff, 1975; Kawashima et al., 2011; Quinlan-Watson, 1953). These results suggest that activation of AHR by an exogenous ligand has the potential to decrease the amount of intracellular zinc and disrupt intracellular zinc homeostasis.
The synthetic flavonoid β-naphthoflavone (5,6-benzoflavone) is another exogenous ligand of AHR (Daujat et al., 1996; Hassoun and Dencker, 1982; Tkachenko et al., 2018) that is non-carcinogenic, unlike the majority of other AHR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene and 3-methylcholanthrene (Burchell and Coughtrie, 1989; Ioannides and Parke, 1987). Thus, this compound is often used as a positive control for AHR activation. In this study, we examined whether AHR activation affects intracellular zinc homeostasis by investigating the effect of β-naphthoflavone on intracellular zinc levels.
Human hepatoma HepG2 cells (three stocks with different lot numbers) were obtained from the Human Science Research Resources Bank (Osaka, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium low glucose (WAKO Pure Chemical Industries, Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells from different stocks were cultured simultaneously and passaged to the same passage number (range 3-10). Prior to treatment, cells were grown in 60-mm culture dishes (AGC Techno Glass Co., Shizuoka, Japan) to 80%–90% confluence. β-Naphthoflavone was purchased from WAKO Pure Chemical Industries, and 0.1, 1, and 10 mM stock solutions of β-naphthoflavone were prepared in dimethyl sulfoxide (DMSO, WAKO Pure Chemical Industries). The concentrations of β-naphthoflavone were based on a previous report (Hanioka et al., 2006). For experiments, HepG2 cells were treated with 0.1% (v/v) β-naphthoflavone stock solution in culture medium for 3 days. Culture medium containing β-naphthoflavone was refreshed every day.
Intracellular labile zinc levels were measured using ZnAF-2 DA (GORYO Chemical, Hokkaido, Japan), a reagent that specifically fluoresces with intracellular labile zinc. ZnAF-2 DA is a diacetylated form of ZnAF-2 and once taken up by cells, it is hydrolyzed to ZnAF-2 (Hirano et al., 2000, 2002). ZnAF-2, which cannot permeate the cell membrane, is a fluorescein-based zinc sensor containing the N,N-bis(2-pyridylmethyl)ethylenediamine chelating unit (Kd = 2.7 nM for Zn2+). β-Naphthoflavone-treated cells were washed with phosphate-buffered saline (PBS, WAKO Pure Chemical Industries) and then incubated for 30 min with medium containing 10 µM ZnAF-2 DA in a CO2 incubator. Cells were harvested in trypsin/EDTA solution (WAKO Pure Chemical Industries) and resuspended in Hank’s balanced salt solution without calcium and magnesium ions (WAKO Pure Chemical Industries). Cells were then stained with 3.75 µM propidium iodide (PI, Dojin Laboratories, Kumamoto, Japan) for 3 min at room temperature, and the fluorescence intensity of ZnAF-2 DA in viable (PI-negative) cells was measured using an Accuri™ C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). Zinc pyrithione (ZPT) and zinc sulfate were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan) and WAKO Pure Chemical Industries, respectively. Stock solutions of 5 mM ZPT and 50 mM zinc sulfate were prepared in DMSO or distilled water, respectively, and added in the medium at 0.1% (v/v) stock solution at 5 min after the start of ZnAF-2 DA treatment. For the measurement of intracellular total zinc levels, cells were collected, washed with PBS, and homogenized by ultrasonic wave. Samples were then diluted 1:2.5 with HNO3 and degraded by microwave. The zinc concentration was determined through inductively coupled plasma atom emission spectroscopy (ICP-AES) using the iCAP 6300 DUO+MFC spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The total zinc content was normalized to the cell number determined by a flow cytometer.
The RNA extraction and qRT-PCR were carried out according to the procedures reported previously (Ishida and Takechi, 2016). Briefly, total RNA was extracted from HepG2 cells using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription was performed using approximately 0.1 µg of total RNA and the ReverTraAce® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). The qRT-PCR assays were performed on a StepOnePlus™ real-time PCR system (Thermo Fisher Scientific) using THUNDERBIRD® SYBR® qPCR Mix (Toyobo) and melting curve analysis. The primer sequences and estimated product sizes have been described previously (Ishida and Takechi, 2016). The Ct value was calculated using StepOnePlus™ software. The relative expression of each target mRNA was determined with reference to β-actin expression.
Significant differences between two groups were calculated using the Student’s t-test. Significant differences among multiple groups were tested by one-way analysis of variance, followed by post hoc multiple comparison test (Tukey–Kramer test).
Exogenous AHR ligands exert their biological effects by altering gene expression through activation of the AHR transcription factor. Cytochrome P450 1A1 (CYP1A1) is a drug-metabolizing enzyme, and is sensitively induced at the transcriptional level through AHR activation. Therefore, the expression level of CYP1A1 mRNA is used as an index of AHR activation. Previous studies have demonstrated that β-naphthoflavone possesses AHR agonist activity and induces CYP1A1 at transcriptional and protein levels (Daujat et al., 1996; Farin et al., 1994; Korashy and El-Kadi, 2004; Raza et al., 1992; Stegeman et al., 1995; Tkachenko et al., 2018; Wang et al., 1997; Yoshinari et al., 2006, 2008). We confirmed that β-naphthoflavone treatment of HepG2 cells for 3 days at 0.1, 1, and 10 µM resulted in a dose-dependent increase of CYP1A1 mRNA levels, with significantly increased levels observed at 1 µM compared with control (Fig. 1).
Effect of β-naphthoflavone treatment on cytochrome P450 1A1 (CYP1A1) mRNA levels. HepG2 cells were treated with the indicated concentrations of β-naphthoflavone for 3 days. The level of CYP1A1 mRNA was determined by qRT-PCR. Data are normalized to β-actin mRNA levels. Values represent the mean ± S.D. of three samples. ***p < 0.001 compared with control (0.1% DMSO) cells.
To clarify whether β-naphthoflavone affects intracellular zinc levels, we examined the level and subcellular distribution of intracellular zinc in β-naphthoflavone-treated cells. The levels of labile zinc in viable cells, which were separated by PI staining, were assessed by the fluorescence intensity of ZnAF-2 DA. As a positive control, we used ZPT, which is a 1:2 complex between a central zinc atom and the membrane permeable ionophore pyrithione (N-hydroxy-2-pyridinethione) (Barnett et al., 1977) that increases intracellular zinc levels (Tuncay et al., 2011). We observed a significant increase of fluorescent intensity in 5 µM ZPT-treated cells, which indicated an increase of intracellular labile zinc levels (Fig. 2A). In comparison, we found that the intracellular labile zinc levels in β-naphthoflavone-treated cells were significantly decreased in a dose-dependent manner (Fig. 2B). The levels of intracellular labile zinc in cells treated with 0.1, 1, and 10 µM β-naphthoflavone were decreased by approximately 5%, 10%, and 17%, respectively, compared with control cells. We also observed a similar trend in the decrease in the level of intracellular total zinc in response to 10 µM β-naphthoflavone, although the difference was not statistically significant (Fig. 3).
Effect of β-naphthoflavone treatment on intracellular labile zinc content. (A) Percentage of intracellular labile zinc levels in control and zinc pyrithione (ZPT)-treated HepG2 cells. Labile zinc in HepG2 cells was measured as the fluorescence intensity of ZnAF-2 DA. ZPT (5 µM) was added to the medium during ZnAF-2 DA treatment. (B) Percentage of intracellular labile zinc levels in control and β-naphthoflavone-treated HepG2 cells. HepG2 cells were treated with β-naphthoflavone at the indicated concentrations for 3 days. Intracellular labile zinc was measured as the fluorescence intensity of ZnAF-2 DA. Values represent the mean ± S.D. of three samples. ***p < 0.001 compared with control (0.1% DMSO) cells.
Effect of β-naphthoflavone treatment on intracellular total zinc content. HepG2 cells were treated with 10 µM β-naphthoflavone for 3 days. Control cells were cultured in medium with 0.1% DMSO. Intracellular total zinc was measured by ICP-AES. Values represent the mean ± S.D. of three samples.
In addition, observation of the subcellular distribution of labile zinc demonstrated that β-naphthoflavone treatment increased the number of intracellular vesicles accumulating labile zinc (Fig. 4).
Intracellular distribution of labile zinc in β-naphthoflavone-treated HepG2 cells. HepG2 cells were treated with 10 µM β-naphthoflavone for 3 days. Intracellular labile zinc was labeled with ZnAF-2 DA (green), and nuclei were stained with Hoechst 33342 (blue). Top: control (0.1% DMSO) treatment. Bottom: 10 µM β-naphthoflavone treatment for 3 days. Bars: 20 µm.
Zinc can be supplied through food, supplementation, and pharmaceutical agents as effective countermeasures against zinc deficiency. We next examined the effect of supplying zinc on the decrease of intracellular labile zinc induced by β-naphthoflavone.
The addition of 5 µM ZPT, which supplies zinc ions into cells as ionophores, after β-naphthoflavone treatment restored the levels of intracellular labile zinc to control levels (Fig. 5A). In contrast, the addition of 50 µM zinc sulfate, which supplies zinc ions into cells through zinc transporters, after β-naphthoflavone treatment was unable to improve the intracellular labile zinc levels altered by β-naphthoflavone (Fig. 5B).
Effect of ZPT and zinc sulfate on the decrease in intracellular labile zinc induced by β-naphthoflavone. (A, B) HepG2 cells were treated with 10 µM β-naphthoflavone for 3 days. Intracellular labile zinc was measured as the fluorescence intensity of ZnAF-2 DA. ZPT (5 µM) or zinc sulfate (ZnSO4, 50 µM) was added to the medium during ZnAF-2 DA treatment. Percentage of intracellular labile zinc levels in cells treated with ZPT and ZnSO4 compared with controls are shown. Values represent the mean ± S.D. of three samples. **p < 0.01, ***p < 0.001 compared with control (0.1% DMSO) cells. †p < 0.05 compared with β-naphthoflavone-treated cells.
Zinc transporters play critical roles in the regulation of the intracellular labile zinc concentration. Zinc transporters are divided into two groups, zinc transporter (ZnT) family proteins and Zrt/Irt-like Protein (ZIP) family proteins. To date, 10 ZnT isoforms (ZnT1–10) and 14 ZIP isoforms (ZIP1–14) have been reported in mammalian cells along with their tissue/cell distributions and subcellular localizations (Huang and Tepaamorndech, 2013; Jeong and Eide, 2013). Thus, to examine the mechanism underlying the decrease in intracellular labile zinc by β-naphthoflavone, we examined the mRNA levels of zinc transporters. Although significant changes in mRNA expressions of the majority of zinc transporters were not detected in response to β-naphthoflavone, the mRNA levels of ZnT10 and ZIP5 were significantly decreased by 10 µM β-naphthoflavone (Fig. 6). The expression levels of ZnT10 and ZIP5 mRNA were approximately 30% and 40%, respectively, of control levels.
Effect of β-naphthoflavone treatment on zinc transporter mRNA levels. HepG2 cells were treated with 10 µM β-naphthoflavone for 3 days. The expression level of each mRNA was determined by qRT-PCR. Data were normalized to β-actin mRNA levels. Controls were set to 1.0, and values represent the mean ± S.D. of three samples. *p < 0.05 compared with control (0.1% DMSO) cells.
Here, we showed that β-naphthoflavone induces a decrease in the level of intracellular labile zinc. We also observed a tendency for β-naphthoflavone (10 µM) to induce a decrease of intracellular total zinc levels, and an alteration of intracellular labile zinc distribution with an increase of the number of vesicles incorporating intracellular labile zinc.
Previous studies have demonstrated that zinc-binding proteins, including carbonic anhydrase III, aldolase B, and alcohol dehydrogenase, were suppressed by an exogenous AHR ligand in rat livers (Ikeda et al., 2000; Ishii et al., 1997, 2001). These results including our observations suggested that exogenous AHR ligands have the potential to decrease intracellular levels of zinc, including both labile and bound forms, and disrupt intracellular zinc homeostasis. Previous reports showed that the AHR ligands dioxins and benzo(a)pyrene can also cause numerous adverse effects in mammals. For example, treating laboratory animals with dioxins can result in many forms of toxicity, including wasting syndrome, hepatotoxicity, and immunosuppression (Poland and Knutson, 1982). Additionally, an epidemiological analysis in humans found that chronic or accidental exposure to low doses of dioxins is a potential risk factor for type 2 diabetes (Bertazzi et al., 2001; Wang et al., 2008) and cancer (Leng et al., 2014; Wang et al., 2013). Furthermore, recent findings have demonstrated a relationship between zinc homeostasis disruption and the onset of disease, such as type 2 diabetes (Chu et al., 2017; Yary et al., 2016), Alzheimer’s disease (Kawahara et al., 2018; Li and Wang, 2016; Portbury and Adlard, 2015), and cancer (Bafaro et al., 2017; Kolenko et al., 2013; Ressnerova et al., 2016). The precise physiological consequence of disrupted zinc homeostasis by AHR ligands remains to be determined. However, we speculate that disruption of zinc homeostasis may be an important adverse effect that can occur with AHR activation. In addition, from the previous reports regarding CYP1A1 gene expression assay and xenobiotic responsive element (XRE)-luciferase reporter gene assay, it is suggested that the effects of 10 µM β-naphthoflavone on mammalian cells were comparable with those at 1 ~ 10 nM TCDD, which is one of most toxic dioxins (Horling et al., 2011; Zordoky and El-Kadi, 2010). Thus, the decrease of intracellular zinc level by β-naphthoflavone that we observed could be induced by the exposure of TCDD at nanomolar concentration. According to the environmental survey for dioxins as of 2017, a public report from Ministry of the Environment, the average concentrations of dioxins in environment were greatly low compared with standard values (air, 0.019 pg-TEQ/m2; Public water quality, 0.17 pg-TEQ/L; Soil, 3.4 pg-TEQ/g). Furthermore, the daily intake of dioxins from food as of 2017 was estimated at 0.65 pg-TEQ/kg body weight/day in the survey of daily intake of dioxins from food, a public report from Ministry of Health. Under present circumstance, the dioxins from environment and food will have little effect on the physiological zinc homeostasis. However, dioxins are recognized as an environmental pollutant that has hardly decomposable and highly accumulating properties in vivo. Therefore, it is unable to deny the possibility that the persistent exposure to dioxins from environment and food may become the future risk factor of the disruption of physiological zinc homeostasis.
To investigate the potential mechanism in the β-naphthoflavone-induced decrease of intracellular zinc level, we measured the mRNA levels of zinc transporters. Our results showed that the mRNA levels of ZnT10 and ZIP5 were significantly decreased in 10 µM β-naphthoflavone-treated cells. ZnT10 is expressed in various tissues with the highest levels of expression in small intestine, liver and brain tissues (Bosomworth et al., 2012). This transporter was initially thought to act as a zinc efflux transporter; however, recent studies suggested that this transporter also functions as a manganese efflux transporter (Quadri et al., 2012; Tuschl et al., 2012). ZIP5 is primarily expressed in the liver, kidney, pancreas and small intestine and plays an important role in maintaining cellular zinc levels by facilitating the uptake of dietary zinc into intestinal epithelial cells and releasing zinc from vesicular compartments (Dufner-Beattie et al., 2004; Wang et al., 2004). While both transporters are localized in the plasma membrane of mammalian cells (Bosomworth et al., 2012; Jeong and Eide, 2013), little is known about the interaction of these transporters. Furthermore, it remains unclear how these two zinc transporter mRNAs were decreased by β-naphthoflavone. Our results demonstrated that the dose dependent manner of the increase of CYP1A1 mRNA by β-naphthoflavone was parallel with that of the decrease of intracellular zinc level by the compound. Therefore, it is most likely that the activation of AHR was involved in the events induced by β-naphthoflavone. However, although a deduced XRE sequence, a responsive element of activated AhR, was found at approximately 3.8 kbp upstream of the start codon of ZnT10, the sequence was not found in upstream region of ZIP5. Thus, although an activation of AHR was start site in the events by β-naphthoflavone, it remains controversial whether the activated AHR signal pathway had directly effect on expression of ZnT10 and ZIP5 mRNAs. One possibility is that other signal transduction induced by AHR activation was involved in the effect of β-naphthoflavone. For example, β-naphthoflavone is reported to activate the nuclear factor erythroid 2-related factor 2 (Nrf-2) signal pathway (Moinova and Mulcahy, 1999). Beside, it is reported that the activation of AHR by exogenous ligands, such as benzo(a)pyrene, 3-methylcholanthrene and β-naphthoflavone, produced the occurrence of oxidative stress (Elbekai et al., 2004). Therefore, we speculate that the activation of other signal pathway (e.g. Nrf-2 signal pathway) following the activation of AHR was involved in the decrease of ZnT10 and ZIP5 mRNAs by β-naphthoflavone. On the other hand, although there was a decrease of intracellular zinc levels in cells treated with β-naphthoflavone, most mRNA levels of zinc transporters involved in an efflux/influx of extracellular labile zinc were not changed. ZIPs regulate zinc ion influx from either the extracellular milieu or intracellular vesicles to increase the cytoplasmic zinc concentration (Kambe et al., 2014; Lichten and Cousins, 2009; Zhao and Eide, 1996), and ZIP5 is localized to the plasma membrane in mammalian cells (Jeong and Eide, 2013). This suggests that ZIP mRNA expression would be induced following a decrease in intracellular zinc levels to supply extracellular zinc ions into the cytoplasm (Dufner-Beattie et al., 2003; Jain et al., 2013; Weaver et al., 2007). However, our results showed that ZIP5 was decreased, while other ZIPs were unaffected in 10 µM β-naphthoflavone-treated cells. Therefore, we speculated that the induction of ZIP mRNAs may be inhibited by transcriptional repression from AHR activation even under conditions in which intracellular zinc levels were decreased, leading to disrupted influx of zinc ions from the extracellular fluid. This hypothesis is supported by our observation that the decrease in intracellular labile zinc levels induced by β-naphthoflavone was not improved by an increase in extracellular zinc ions derived from zinc sulfate. In this study, we examined mRNA levels at 24 hr after last β-naphthoflavone exposure and thus cannot exclude the possibility of changes in zinc transporter mRNA expressions in earlier stages of β-naphthoflavone exposure. The precise mechanism by which activation of AHR by β-naphthoflavone treatment resulted in decreased intracellular zinc level remains unknown and further studies are needed to clarify these issues.
In conclusion, our results demonstrated that intracellular zinc levels in HepG2 cells were impacted by β-naphthoflavone. Because β-naphthoflavone is an exogenous ligand of the AHR, our study suggests that activation of the AHR by exogenous ligands, such as dioxins and benzo(a)pyrene, has the potential to disrupt intracellular zinc homeostasis. Although the mechanism remains to be fully clarified, our data provide preliminary evidence that suppression of the transcription of several zinc transporters may be involved. The mechanisms underlying the adverse effects produced by exogenous AHR ligands require further exploration. However, the concept of zinc homeostasis disruption in the adverse effects by exogenous AHR ligands provides a new approach for further investigation.
We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
The authors declare that there is no conflict of interest.