TCDD promotes liver fibrosis through disordering systemic and hepatic iron homeostasis

Journal of Hazardous Materials 395 (2020) 122588

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TCDD promotes liver fibrosis through disordering systemic and hepatic iron homeostasis

T

Changying Lia,b, Yingying Liua, Zheng Dongb,c, Ming Xub,c, Ming Gaob,c,**, Min Conga,*, Sijin Liub,c a

Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis and National Clinical Research Center of Digestive Diseases, Beijing, 100050, China b State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China c University of Chinese Academy of Sciences, Beijing, 100049, China

GRAPHICAL ABSTRACT

ARTICLE INFO

ABSTRACT

Editor: D. Aga

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic environmental pollutant which can cause severe health problems, such as fibrosis. However, the toxic effects and related mechanism of TCDD on the liver remain largely unknown. In this study, we established a liver fibrosis mouse model upon exposure of TCDD, as evidenced by increased collagen I, tumor growth factor β1 (TGFβ1), α-smooth muscle actin (α-SMA), and Masson staining. Meanwhile, there was also a significant increase of inflammatory factors and TUNEL-positive hepatocytes in liver, indicating that liver inflammation and hepatic cell apoptosis occurred. In addition, increased serum and liver iron were concomitant with liver injury induced by TCDD. We further investigated the mechanism

Keywords: TCDD Liver fibrosis Cideb Hepatocyte apoptosis Iron deposition

Abbreviations: α-SMA, α-smooth muscle actin; AHR, aryl hydrocarbon receptor; Akt, protein kinase A; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Bax, BCL-2-associated X protein; Chk1, checkpoint kinase 1; CIDE, cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector; DMSO, dimethyl sulfoxide; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; Fasl, Fas ligand; Fe-NTA, ferric nitrilotriacetate; HAMP-/-, hepcidin knockout; HSC, hepatic stellate cell; IHC, immunohistochemistry; IL-1β, interleukin-1β; IL-6, interleukin-6; NF-κB, nuclear factor-κB; Nme5, NME/NM23 family member 5; Gadd45α, recombinant growth arrest and DNA damage inducible protein alpha; qPCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; TCDD, 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin; TGFβ1, tumor growth factor β1; TLR9, toll-like receptor 9; TNFα, tumor necrosis factor alpha; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ⁎ Corresponding author. ⁎⁎ Corresponding author at: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China. E-mail addresses: [email protected] (M. Gao), [email protected] (M. Cong). https://doi.org/10.1016/j.jhazmat.2020.122588 Received 10 October 2019; Received in revised form 19 March 2020; Accepted 24 March 2020 Available online 07 April 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.

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underlying TCDD-induced hepatocyte apoptosis through apoptosis polymerase chain reaction array, and found that a crucial apoptosis-related gene, cell death-inducing DFF45-like effector b (Cideb), was significantly increased in primary hepatocytes from TCDD-exposed mice, and accompanied by liver iron deposition in hepcidin knockout mice. Therefore, Cideb depletion could effectively attenuated TCDD or iron induced cell death related genes expression. In conclusion, our results showed that iron-induced Cideb expression played a critical role in promoting TCDD-induced hepatocyte apoptosis and liver fibrosis, which provide a novel mechanism for understanding TCDD-induced liver injury.

1. Introduction

2. Materials and methods

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD), a kind of persistent organic pollutants, is a toxic environmental contaminant which often accumulates in the environment due to its high concentrations and lower elimination. Several studies have shown that TCDD may cause adverse health effects in humans (Pelclova et al., 2006). It can be transferred via eating, drinking, and touching contaminated substances, subsequently absorbed, and metabolized by biological tissues, leading to disorders of the systemic metabolic, endocrine, and immune system functions (Mori and Todaka, 2017). An increasing number of studies have indicated that TCDD generates the spectrum of biological effects and dioxin-like toxicity in organs and systems (Volz et al., 2005), especially the liver (Watson et al., 2014; Czepiel et al., 2010; Angrish et al., 2013). The changes caused by TCDD in the liver are hepatocyte edema, disordered liver structure, macrophage polarization (Wang et al., 2015), and tumor formation (Ray and Swanson, 2009; Viluksela et al., 2000; Dragan and Schrenk, 2000). Liver fibrosis is a dynamic process of extracellular matrix (ECM) formation and degradation which could further progress to liver cirrhosis and even hepatocellular carcinoma (Schuppan, 2000). Briefly, apoptotic hepatocytes upon liver injury could form apoptosis bodies and then recruit inflammatory cells, such as macrophages, to gather in the damaged area of liver to secrete inflammatory cytokines, which finally promote the activation of hepatic stellate cell (HSC) (Schuppan, 2000; Weiskirchen and Tacke, 2016). Exposure to environmental pollutants, such as heavy metals and TCDD, have been reported to increase the potential risk for liver fibrosis (Han et al., 2017; Harvey et al., 2016). A recent study showed that HAMP (the encoding gene of hepcidin) repression and iron accumulation exacerbated the progression of TCDDelicited hepatotoxicity through the mechanism of oxidative stress (Fader et al., 2017). It was reported that iron overload could cause Fenton reaction in hepatocytes and macrophages, which lead to the progression of liver fibrosis (Mehta et al., 2019). In addition, iron could also stimulate HSC and Kupffer cell activation, which promotes the secretion of pro-inflammatory cytokines and liver fibrosis progression (Wood et al., 2014). Furthermore, iron overload could induce fibrogenic signal in parenchymal and non-parenchymal cells, consequently inducing the progression of liver fibrosis. Considerable evidence has shown that TCDD could cause hepatotoxicity and DNA damage by activating the main drug metabolism enzyme cytochrome P450/AHR (aryl hydrocarbon receptor) in the liver (Chen et al., 2004; Cantrell et al., 1996). However, the key regulator of hepatotoxic and liver fibrosis induced by TCDD was still largely unknown. Cideb, namely cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector b, is mainly expressed in liver and promotes cell apoptosis dependent on caspase families, such as caspase-3, caspase-9, and cytochrome c (Erdtmann et al., 2003; Liu et al., 2009; Slayton et al., 2019). Here, our results showed that TCDD could cause liver fibrosis by promoting hepatocyte apoptosis through upregulating iron-induced Cideb expression, which provides a new aspect to evaluate the health risks of TCDD and provides new insights into the mechanism of TCDD-induced hepatotoxicity and liver fibrosis.

2.1. Animals and TCDD treatment Eight-week-old male Balb/C mice were purchased from Vital River Laboratories, and mice were raised in the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences. Mice were bred at a specific pathogen-free facility. Prior to experiments, all animal experimental protocols were approved by the Animal Ethics Committee at the Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. All animals were housed at comfortable temperature and humidity (60 ± 5%) under 12 h light-dark cycle. TCDD was dissolved in dimethyl sulfoxide (DMSO, Solarbio, China) after nitrogen purging at stock concentrations of 100 mg/mL and then diluted 20 times and 200 times in corn oil (Solarbio, China). After an adaptive phase of 3 days, 72 male Balb/C mice were divided randomly into 12 groups (n = 6), namely mice injected intra-peritoneally with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight once a week for 1, 4, and 6 weeks. Eight- and 28-week-old male C57 mice, which were purchased from the Vital River of Laboratories, and 8- and 28-week-old male HAMP−/− mice, which were gifted from professor Sophie Vaulont of the National Center for Scientific Research of France and professor Tomas Ganz of the University of California, USA, were used for detecting iron levels in the liver and the expression of Cideb in livers and isolated hepatocytes. 2.2. ELISA (enzyme-linked immunosorbent assay) ELISA kits of serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were purchased from Qiao Du (Shanghai, China). Mouse IL-6 (interleukin-6), IL-1β (interleukin-1β), and TNFα (tumor necrosis factor alpha) ELISA kits were purchased from R&D SYSTEMS. All the experiments were carried out according to the manufacturer′s instructions. The optical density was read using a microplate fluorescence reader (Thermo Fisher, USA). 2.3. Immunohistochemistry (IHC), TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling), and Prussian Blue staining The right lobe livers were fixed with 4 % paraformaldehyde, and the IHC of Masson staining, α-SMA (α-smooth muscle actin), collagen I, F4/ 80, CD45, and TUNEL staining were performed. Images of 5 random microscope fields of staining in each liver section were recorded to quantitative analysis using ImageJ software. Hepatic iron staining was performed by Prussian Blue, and then we used Enhanced Coloring Agent (Solarbio, Beijing, China) to enhance the results. 2.4. Assay for hepatic iron quantitative analysis Mouse livers were broken in a Scientz-48 tissue grinder (Ningbo, China) with beads, and were then incubated with the acid solution (49.6 % HCl, 20 % saturated trichloroacetic acid, 30.4 % H2O, v/v/v) at 65 °C for 72 h. Iron concentration was measured by Chromagen solution as in previous studies (Liu et al., 2019). The optical density was read at

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535 nm with a microplate fluorescence reader (Thermo Fisher, USA).

contrast, when under industrial accident such as Seveso disaster, the median lipid-adjusted serum TCDD levels in residents within high contaminated territory rose to 73.3 ppt and 12.4 ppt, extremely higher than that in the populations who lived in low contaminated zones (around 5.5 ppt) (Consonni et al., 2012). Recently, a study indicated that serum TCDD concentrations in populations who worked in a New Zealand phenoxy herbicide production plant manifested a mean TCDD lipid-adjusted serum concentration around 19.1 ppt (t Mannetje et al., 2016). Moreover, chlorophenol workers harbored higher TCDD level than workers without chlorophenol exposures, and the median lipidadjusted serum TCDD level was 30.5 ppt in trichlorophenol workers with chloracne (Collins et al., 2006). All these previous studies revealed that the concentrations of 1–30 ppt could literately reflect the levels of TCDD in the real exposure scenarios with significant environmental relevance. Based on the dose conversion method, the equivalent dose of TCDD for humans relative to mice is about 1:12.3 (Nair and Jacob, 2016). According to the physiologically pharmacokinetic models (Emond et al., 2005), exposure in mice upon 5 μg/kg TCDD is subjected to a concentration of 67 ppt in blood. Therefore, the lipid-adjusted blood concentration of TCDD in mice ranged from 13.4 to 335 ppt post administration of TCDD at 1−25 μg/kg body weight, in parallel to 1.08–27.2 ppt in humans. Therefore, to better imitate the realistic environmental health and safety, we chose the dosage of 1−25 μg/kg body weight of TCDD in mice. To establish a TCDD-induced liver fibrosis mouse model, we fed male Balb/C mice with 1, 10, or 25 μg/kg TCDD or the vehicle (DMSO) for 1, 4, or 6 weeks, and then the deposition of ECM and the activation of HSC in the liver in TCDD-treated mice were analyzed by qPCR (quantitative real-time RT-PCR) and IHC. As shown in Fig. 1A–C and Supplemental Fig. 1A–D, the mRNA and protein expression levels of collagen I, TGFβ1 (tumor growth factor β1) and α-SMA were all significantly increased in TCDD-exposed mice in dose- and time-dependent manner. In addition, Masson staining results showed more severe liver fibrosis in TCDD-exposed mice in a dose-dependent manner. In particular, 25 μg/kg TCDD-fed mice displayed severe bridge fibrosis at 6 weeks (Fig. 1D, Supplemental Fig. 1E), indicating that chronic TCDD

2.5. Quantitative RT-PCR The total RNA of the liver was isolated using Trizol reagent (Invitrogen, USA), and first strand cDNA was synthesized using a Thermo script™ RT-PCR system (Thermo scientific, USA) according to the manufacturer′s instructions. cDNA was performed to PCR cycles by using the SYBR green quantitative PCR reagent (Promega), and assays were performed using the CFX96 Real-Time System (Bio-Rad). The primers are shown in Table 1. 2.6. Apoptosis PCR array Primary hepatocytes from TCDD-exposed Balb/C mice of different doses for 1 week and HAMP−/− mice were digested with enzymes in portal vein perfusion as previously described (Seki et al., 2007). Primary hepatocytes from TCDD-exposed Balb/C mice were subjected to Apoptosis PCR array (Qiagen). The experiment was carried out according to the manufacturer′s instructions, and assays were performed by using the CFX96 Real-Time System (Bio-Rad). Primary hepatocytes from HAMP−/− mice were examined for mRNA expression of Cideb. 2.7. Western blotting Cells were harvested and lysed using radioimmunoprecipitation assay buffer (Solarbio, Beijing, China) containing protease inhibitor cocktail (Roche, Switzerland). The experiment was carried out as in previous studies (Gao et al., 2018). We incubated blots with antibody against Nme5 (Proteintech, Chicago, USA), Cideb (Proteintech, Chicago, USA) and β-actin (Santa Cruz) and then visualized them using the enhanced chemiluminescence light method. 2.8. Colony formation assay and cell death assay The NCTC 1469 cells were seeded into 6-well dishes at a density of 1 × 103/well in the presence of either the vehicle (1% DMSO) or TCDD (0.1, 10, 20, 50, 80, 100 nM) for 10 days. The experiment was carried out as in previous studies (Yamaguchi and Hankinson, 2018). The colonies were counted with Image J. The NCTC 1469 cells were seeded into 6-well dishes at a density of 1 × 105/well in the presence of TCDD (100 nM) for 24 h or ferric nitrilotriacetate (Fe-NTA) (1.5 mM) for 4 h with or without Cideb siRNA (sense: CUAAGAUCUCAGCUUUAUATT, antisense: UAUAAAGCUGAG-AUCUUAGCC)(Jima, Suzhou, China). FeNTA was composed of 0.1 mol/L FeNO3 (Sigma, USA), 0.1 mol/L Na2NAC (Fluka, USA) and NaHCO3, which adjusted to pH 7.4 and filtered with 0.22 μmol/L millipore filter.

Table 1 PCR primers used in this study. Gene

Primer sequences

Cyclophilin

F-GGAGATGGCACAGGAGGA R-GCCCGTAGTGCTTCAGCTT F-ATGGAGGGGAATACAGCCC R-TTCTTTGCAGCTCCTTCGTT F-TAGGCCATTGTGTATGCAGC R-ACATGTTCAGCTTTGTGGACC F-GTTCAGTGGTGCCTCTGTCA R-ACTGGGACGACATGGAAAAG F-TGGAGCAACATGTGGAACTCT R-CCTGTATTCCGTCTCCTTGGT F-CTGCAAGAGACTTCCATCCAG R-AGTGGTATAGACAGGTCTGTTGG F-GGTCAAAGGTTTGGAAGCAG R-TGTGAAATGCCACCTTTTGA F-AGGGTCTGGGCCATAGAACT R-CCACCACGCTCTTCTGTCTAC F-CTGAGCAGCACCACCTATCTC R-TGGCTCTAGGCTATGTTTTGC F-TCCGTGAGTTCACCAACCAAA R-GGGGGTTCCCTGTTAAATGGG F-TCAGGGCTAGGACACCG R-TTGAAGATGAGGGCAACTCC F-TGAAGACAGGGGCCTTTTTG R-AATTCGCCGGAGACACTCG F-CAGGTTCATGTTTCCAGCCG R-TCCTTGAAGTAGGGTTGGCG F-GCTCCAATGGCCTGCTAAG R-TTATGATCACAGACACGGAAGG

β-actin Collagen I α-SMA

2.9. Statistical analysis

TGFβ1

Statistical analyses were performed using the Student's t-test, oneway analysis of variance, and Pearson correlation. Results are expressed as mean ± standard error of the mean for three independent experiments. All data were analyzed using SPSS 19.0 statistical software. Pvalue less than 0.05 (*: P < 0.05) or 0.001 (**: P < 0.001) indicated statistic difference.

IL-6 IL-1β TNFα Hepcidin

3. Results and discussion

Fasl Casp6

3.1. Chronic TCDD treatment induced liver fibrosis in mice

Bax

A wealth of epidemiological evidence suggests that individuals exposed to numerous organic pollutants have much higher blood TCDD levels (Marques and Domingo, 2019). It was documented that the average lipid-adjusted serum TCDD level in individuals who lived in TCDD poisoning area was approximately 2 ppt (Sorg et al., 2009). By

Nme5 Cideb

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Fig. 1. TCDD induced liver fibrosis. Changes of the mRNA expression of collagen I (A) in livers from 8-week-old Balb/C mice treated with TCDD or vehicle control (DMSO) at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Changes of the mRNA expression of TGFβ1 (B) in livers from 8-week-old Balb/C mice treated with TCDD or vehicle control (DMSO) at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Changes of the mRNA expression of α-SMA (C) in livers from 8-week-old Balb/C mice treated with TCDD or vehicle control (DMSO) at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Changes of Masson staining (D, 200×) in livers from 8-week-old Balb/C mice treated with TCDD or vehicle control (DMSO) at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). *: P < 0.05; **: P < 0.01, relative to DMSO control.

could effectively promote liver fibrosis in mice, analogous to previous studies (Han et al., 2017; Harvey et al., 2016).

3.3. TCDD induced hepatic cell apoptosis in the early stage of liver fibrosis Several studies have found that hepatocyte apoptosis is the main driver of chronic liver inflammation and fibrosis triggered by viral infection, steatosis, and alcohol (Cong et al., 2018). In our study, we found that the serum ALT and AST (Supplemental Fig. 2A and B) were increased in TCDD-exposed mice compared to control mice, indicating that TCDD could cause liver injury possibly due to hepatocyte apoptosis. Several research groups also announced that TCDD could promote cell apoptosis through immunotoxicity (Chopra and Schrenk, 2011). For example, one article underlined that TCDD could promote endothelial apoptosis through the COX‐2/PGE2/EP3/p38/Bcl‐2 pathway (Yu et al., 2017); another study reported that mouse embryonic fibroblasts from AHR-null mice were more sensitive to TCDD and exhibit high levels of apoptosis through upregulating TGFβ1 level (Elizondo et al., 2000). Therefore, we hypothesized that TCDD-induced hepatocyte apoptosis might trigger liver inflammation and subsequently liver fibrosis. As shown in the TUNEL staining results (Fig. 3A and B), 10 μg/ kg and 25 μg/kg of TCDD administration for 1 week could cause more hepatic cell apoptosis, indicating that TCDD could promote hepatocyte apoptosis in the early stage of liver fibrosis. Mounting evidences have shown that hepatocyte apoptosis acts as an initiating factor for regulating liver fibrosis. Apoptotic hepatocyte bodies can activate

3.2. TCDD stimulated inflammation in the liver of TCDD-treated mice Previous studies have shown that inflammation was indispensable for the progression of liver fibrosis (Cong et al., 2017). Therefore, we detected the infiltration of inflammatory cells and the expression levels of inflammatory factors to verify the inflammation in TCDD-induced liver fibrosis. As shown in Fig. 2A–D, the F4/80 and CD45 positive inflammatory cells, especially macrophages and leukocytes, were markedly accumulated in the liver of TCDD-exposed mice. In addition, TCDD could also enhance the mRNA expression and serum levels of IL6, IL-1β, and TNFα (Fig. 2E–J), indicating that TCDD could stimulate liver inflammation accompanied with liver fibrosis. Studies have shown that TCDD could induce HSC activation via activating the Akt (protein kinase A) and NF-κB (nuclear factor-κB) signaling pathways (Han et al., 2017; Harvey et al., 2016). IL-6, IL-1β, and TNFα are all downstream targets of Akt and NF-κB signaling pathways; thus, we suppose that TCDD might stimulate liver inflammation through activation of Aktand NF-κB-governed signaling networks, which will be confirmed in a future study. 4

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Fig. 2. TCDD stimulated liver fibrosis accompanied liver inflammation and the secretion inflammatory factors. Changes of F4/80 in IHC (A) and quantitative analysis of the F4/80 positive area (B) in livers from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). CD45 in IHC (C) and quantitative analysis of the CD45 positive area (D) in livers from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Serum IL-6 (E) and the mRNA expression of IL-6 in livers (F) from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Serum IL-1β (G) and the mRNA expression of IL-1β in livers (H) from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Serum TNFα (I) and the mRNA expression of TNFα in livers (J) from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). *: P < 0.05; **: P < 0.01, relative to DMSO control.

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Fig. 3. TCDD led to hepatic cell apoptosis in early stage of liver fibrosis. Changes of TUNEL staining (A) in livers from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1 week (n = 6). Changes of quantitative analysis of apoptotic cells (B) in livers from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1 week (n = 6).

Fig. 4. TCDD-induced iron disorder. Levels of hepatic iron (A) from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Levels of serum iron (B) from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, 6 weeks (n = 6). Levels of the hepcidin mRNA expression (C) in livers from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). Prussian Blue stainging with DAB (D) in livers from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight at 1, 4, and 6 weeks (n = 6). *: P < 0.05; **: P < 0.01, relative to DMSO control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 6

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tissue damage and cell death (Starke and Farber, 1985). It has been reported that iron deficiency could decrease TCDD-induced hepatotoxicity (Sweeny et al., 1979), whereas iron overload could induce liver injury via non-transferrin bound iron and iron deposits (Brissot et al., 2012; Thompson and Bruick, 2012). Therefore, we speculated that iron deposition after TCDD exposure might promote hepatocyte apoptosis. In our study, we found that TCDD could increase iron accumulation in mouse livers, which is consistent with previous studies (Fader et al., 2017). As shown in Fig. 4A and B, hepatic iron and serum iron were significantly increased in TCDD-exposed mice in a dose-dependent manner and with a mounting pattern over the time course. Moreover, hepatic iron accumulated in hepatocytes in livers from long-term TCDD-exposed mice (Fig. 4D), indicating that iron overload might contribute to TCDD-induced hepatocyte apoptosis. The mechanisms that how iron-loaded hepatocytes affect fibrosis initiation and progression have been carefully studied. Excess iron in hepatocytes can feed the Fenton reaction that generates noxious reactive oxygen species (ROS), and then ROS-induced lipid peroxidation of cellular membranes contributes to hepatocyte apoptosis and necrosis (Mehta et al., 2019). In addition, iron-loading cells collectively produce elevated levels of proliferative, proinflammatory and profibrogenic mediators including TGF-β, which ensures a static transformation of HSC to myofibroblasts (Philippe et al., 2007). To interrogate the mechanism whereby TCDD caused the accumulation of iron in hepatocytes, we detected the level of hepcidin, a master regulator of systemic iron homeostasis. Hepcidin functions to compromise the serum iron level through binding and inducing the internalization and degradation of its receptor, frerroportin, a transmembrane iron exporter universally expressed in most types of cells, including macrophages, hepatocytes, and the basal membrane of duodenal enterocytes (Liu et al., 2016). As described previously, TCDD repressed hepcidin (HAMP) gene transcription by diminishing the levels of “iron sensing” complex, and thereby limiting BMP6-SMAD-driven HAMP expression (Fader et al., 2017). Thus, it could be concluded that TCDD, at least partially, alters systemic iron distribution profiles through regulating the expression of hepcidin. As shown in Fig. 4C, the expression level of hepatic hepcidin remarkably declined in response to TCDD in a dose- and time-dependent manner. Collectively, these data indicated that the TCDD-induced hepcidin reduction would facilitate iron efflux from hepatocytes and Kupffer cells into circulation, in agreement with elevated iron in serum and liver.

Table 2 Differentially expressed genes by Apoptosis PCR array in isolated hepatocytes from WT mice and HAMP−/− mice. Gene

Description

Tnf Tnfrsf10b/11b Cideb

Tumor necrosis factor Tumor necrosis factor receptor superfamily, member 10b/11b Cell death-inducing DNA fragmentation factor, alpha subunitlike effector B Baculoviral IAP repeat-containing 5 Apoptosis inhibitor 5 Activating transcription factor 5 BCL2-associated athanogene 3 BCL2-like 1/BCL2-like 2 BCL2/adenovirus E1B interacting protein 3 Nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105 NME/NM23 family member 5 Caspase3/6 Apoptosis-inducing factor, mitochondrion-associated 1 BCL2-associated X protein BH3 interacting domain death agonist Caspase recruitment domain family, member 10 CD40 antigen DNA fragmentation factor, beta subunit Diablo, IAP-binding mitochondrial protein TNF receptor-associated factor 1 Protein phosphatase 1, regulatory subunit 13B

Birc5 Api5 Atf5 Bag3 Bcl2l1/Bcl2l2 Bnip3 Nfkb1 Nme5 Casp3/6 Aifm1 Bax Bid Card10 Cd40 Dffb Diablo Traf1 Trp53bp2

quiescent HSC and Kupffer cells, and these activated cell populations in turn promote inflammation and fibrogenisis (Cong et al., 2018). Apoptosis of liver cells provoked hepatic inflammation in vivo through activating Fas, caspase-3 cleavage and nuclear translocation of activator protein-1 (Jaeschke, 2002). Liver injury-induced apoptotic hepatocyte could be phagocytosed by HSC, resulting in increased nicotinamide adenine dinucleotide phosphate oxidase and cell survival (Zhan et al., 2006; Jiang et al., 2009). In addition, apoptotic hepatocyte DNA could interact with Toll-like receptor 9 (TLR9) on HSC, which leads to the activation of TLR9 and increased HSC migration (Watanabe et al., 2007). TCDD (0.1–10 nM) has been reported to suppress liver cancer cell HepG2 growth and colony formation via inhibiting cell proliferation and enhancing cell apoptosis (Yamaguchi and Hankinson, 2018). We then questioned whether TCDD could also suppress the cell colony formation ability of hepatocyte line NCTC 1469 cells. As shown in Supplemental Fig. 2C, the NCTC 1469 cells were cultured in the presence of TCDD (0.1, 10, 20, 50, 80, 100 nM) for 10 days, and then colonized cells were detected by quantitative analysis. Our results showed that TCDD could inhibit the colony formation of hepatocyte line NCTC 1469 when treated with relatively low doses of TCDD, further indicating that TCDD promotes hepatocyte apoptosis, although other studies have also shown that TCDD could inhibit cell apoptosis through a mode of tumor promotion (Chopra and Schrenk, 2011). For example, relative high doses of TCDD (1–25 nM) could inhibit hepatocyte apoptosis triggered by chemicals or ultraviolet (Chopra and Schrenk, 2011) and TCDD could inhibit hepatocyte apoptosis via the repression of p53 functions (Besteman et al., 2007; Paajarvi et al., 2005). In addition, TCDD served as an anti-apoptosis agent to increase the production of transforming growth factor alpha in an AHR-dependent manner (Davis et al., 2001). Therefore, whether TCDD exerts proapoptotic or anti-apoptotic effects depends on the exposure dosage, time, and the microenvironment of the cells or tissues.

3.5. TCDD promoted hepatocyte apoptosis through Cideb To elucidate the mechanism by which TCDD could induce hepatocyte apoptosis, we isolated hepatocytes from 1-week TCDD-exposed mice for apoptosis PCR array. This assay kit consists of 84 genes which are involved in the regulation of hepatocyte apoptosis. As shown in Table 2 and Fig. 5A, among of these differentially expressed genes, 24 genes were changed at different degrees upon TCDD treatment. To further identify which candidate is most important for mediating TCDD-induced hepatocyte apoptosis, we used qPCR assay to verify the accuracy and specificity of these genes. As shown in Fig. 5B, there was a close relevance between the results from apoptosis PCR array and qRTPCR (r = 0.762). In addition, the mRNA expression levels of Fasl (Fas ligand), caspase 6, Bax (BCL-2-associated X protein), Cideb, and Nme5 (NME/NM23 family member 5) were all increased in primary hepatocytes isolated from TCDD-exposed mice compared to control mice (Fig. 5C–G). Fasl is a member of the TNF-receptor superfamily. Caspase 6 is a major effector of neuronal cell death and a major protein involved in amyloid precursor protein lysis. Bax is a classically apoptotic molecule, and its elevation indicates that TCDD could promote hepatocyte apoptosis (Schuppan, 2000; Weiskirchen and Tacke, 2016). Nme5, a member of the NME family, could translocate into the nucleus to join in DNA repair when exposed to stimulation, such as DNA damage (Puts et al., 2018). Among them, Nme5 and Cideb could be induced by TCDD

3.4. Iron accumulated in liver of TCDD-treated mice Iron is one of the basic elements of the human body and is involved in the synthesis of hemoglobin, myoglobin, cytochromes and many enzymes. Ferric (valence) iron catalyzes the Haber-Weiss reaction, converting hydrogen peroxide to reactive oxygen species, resulting in 7

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Fig. 5. Cideb increased in liver fibrosis induced by TCDD. A: All genes in the apoptosis PCR array. Group 1 represents death domain receptor, Group 2 represents DNA damage, Group 3 represents caspase inhibitors, Group 4 represents anti-apoptosis: Bax, Bcl2l10, Birc5, Cflar, Dapk1, Tnf, and Trp63 are also in groups. Group 5 represents DEATH domain proteins: Cradd, Fadd, Tnfrsf10b, and Nfkb1 are also in groups. Group 6 represents the extracellular signals. Group 7 represents caspases: Cradd is in groups. Group 8 represents caspases activators: Casp1, Casp9, Tnfrsf10b, and Trp53 are in groups. Group 9 represents other regulator of apoptosis. Correlation between PCR array folds and qPCR folds (B) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, 25 and μg/kg of body weight for 1 week (n = 6). Changes of the mRNA expression of Fasl (C) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight for 1 week (n = 6). Changes of the mRNA expression of Caspase 6 (D) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg body weight for 1 week (n = 6). Changes of the mRNA expression of Bax (E) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg body weight for 1 week (n = 6). Changes of the mRNA expression of Cideb (F) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight for 1 week (n = 6). Changes of the mRNA expression of Nme5 (G) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight for 1 week (n = 6). Changes of the protein expression of Nme5 and Cideb (H) in primary hepatocytes from 8-week-old Balb/C mice treated with TCDD or DMSO at 1, 10, and 25 μg/kg of body weight for 1 week (n = 6). Arrows represent increasing.

even at a low dosage. Therefore, we further verified the protein levels of Nme5 and Cideb in primary hepatocytes from TCDD-exposed mice and found that there was more induction of Cideb than Nme5 in hepatocytes treated with a low dose of TCDD (Fig. 5H). In addition, Cideb has been found to promote DNA fragments in a caspase-dependent mechanism (Inohara et al., 1998), suggesting the Cideb is mainly responsible for TCDD-induced hepatocyte apoptosis.

of iron content and Cideb expression in the liver in 8- and 28-week-old WT mice. However, Cideb expression levels were significantly increased accompanied with incremental liver iron deposition in 28-week-old HAMP−/− mice compared to that in 8-week-old HAMP−/− mice. In addition, the expression of Cideb in hepatocytes isolated from 28-weekold HAMP−/− mice was significantly higher than that in hepatocytes isolated from 8-week-old HAMP−/− mice, indicating that the expression of Cideb in hepatocytes was due to the deposition of liver iron (Fig. 6C). We further utilized siRNA to knockdown Cideb in NCTC 1469 to investigate whether Cideb is mainly responsible for TCDD/iron induced hepatocyte apoptosis. We found that Cideb depletion could effectively attenuate TCDD or iron induced cell death related genes (TNFα, Bax, Fasl, checkpoint kinase 1 (Chk1), recombinant growth arrest and DNA damage inducible protein alpha (Gadd45α)) expression,

3.6. Iron overload-induced Cideb expression To further confirm whether iron accumulation could affect TCDDinduced Cideb expression, the expression levels of Cideb in the livers from 8- and 28-week-old WT and hepcidin knockout (HAMP−/−) mice were detected. As shown in Fig. 6A and B, there was almost no change 8

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Fig. 6. Iron increased Cideb expression and Cideb protected against TCDD or iron induced hepatocyte apoptosis. Changes of Prussian Blue (A) in livers from 8- and 28-week-old C57 and HAMP−/−mice; Changes of the mRNA expression of Cideb (B) in livers from 8- and 28-week-old C57 and HAMP−/−mice; The mRNA expression of Cideb (C) in primary hepatocytes from 8- and 28 -week-old HAMP−/− mice *: P < 0.05, relative to 8-week-old HAMP-/- mice. The mRNA expression of Cideb after treated with or without Cideb siRNA (D); The mRNA expression of TNFα, Bax, Fasl, Chk1, Gradd45α after 100 nM TCDD (E) or 1.5 mM Fe-NTA (F) stimulation with or without Cideb siRNA in NCTC 1469. *: P < 0.05; **: P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

indicating that Cideb could regulate hepatocyte apoptosis induced by TCDD or iron (Fig. 6D–F). In conclusion, the above results demonstrated that iron deposition could increase the expression of Cideb, which in turn leads to the apoptosis of hepatocytes.

4. Conclusions TCDD, due to its high fat-solubility and easy accumulation in the adipose tissue, could cause potential damage to internal organs, 9

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported under grants from the National Natural Science Foundation of China (grant numbers: 21637004, 21920102007 and 81570542), the Beijing Natural Science Foundation (grant number: 8191002 and 7142043) and the international collaboration key grant from the Chinese Academy of Sciences (grant number: 121311KYSB20190010). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.122588. References

Fig. 7. The mechanisms of TCDD-induced liver fibrosis. Liver fibrosis induced by TCDD was a cumulative response involving multiple mechanisms: iron excess, Cideb expression, hepatocyte apoptosis, and inflammation, which finally leads to HSC activation and TCDD-induced liver fibrosis.

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particularly adipose tissue and the liver. Increasing evidence has shown that TCDD could contribute to the progression of liver fibrosis, partially dependent on the activation of the AHR, which leads to lipid and carbohydrate metabolism dysfunctions in the liver. Although it was reported that TCDD directly induced DNA damage and apoptosis mediated by P53 through the activation of cytochrome P450/AHR receptor (Das et al., 2017), whether other mechanisms mediate TCDD-induced hepatocytes apoptosis and liver fibrosis require further investigation. Here, we reported a remarkable increase in inflammation, hepatic cell apoptosis, and hepatic iron deposition, which regulated the progression of TCDD-induced liver fibrosis. We further found that a crucial apoptosis-related gene, Cideb, was significantly increased in isolated primary hepatocytes from TCDD-exposed mice. In vitro and in vivo results showed that the increase of Cideb was due to increased serum and liver iron, as a result of TCDD-induced liver injury. In addition, Cideb depletion could effectively attenuate TCDD or iron induced cell death related genes expression. In conclusion, our study showed that liver fibrosis induced by TCDD was a cumulative response involving multiple mechanisms: iron excess, Cideb expression, hepatocyte apoptosis, and inflammation, which finally leads to HSC activation and TCDD-induced liver fibrosis (Fig. 7). Our finding signified that iron accumulation could promote hepatocyte apoptosis through Cideb in response to TCDD, which enhances our understanding of TCDD-induced hepatocyte apoptosis and liver fibrosis. Author contributions All listed authors participated meaningfully in the study and have read and approved the submission of this manuscript. C.L., Y.L., Z.D., and M.X. performed the research and collected the data. M.C. and M.G. established the hypotheses, supervised the studies, analyzed the data, and co-wrote the manuscript. S.L. provided the experimental platform and technical support. The authors thank the laboratory members of the Research Center for Eco-Environmental Sciences of the Chinese Academy of Sciences for reagents and assistance with experiments.

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