Triclocarban triggers osteoarthritis via DNMT1-mediated epigenetic modification and suppression of COL2A in cartilage tissues

doi:10.1016/j.jhazmat.2023.130747

Journal of Hazardous Materials
有害物质杂志

Volume 447, 5 April 2023, 130747
第447卷，2023年4月5日，130747

https://doi.org/10.1016/j.jhazmat.2023.130747 Get rights and content 获取权限和内容

Under a Creative Commons license
根据知识共享许可

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Highlights 突出

•
Triclocarban triggers osteoarthritis.
三氯卡班会引发骨关节炎。
•
Triclocarban reduces the cartilage tissue to narrow the intraarticular space.
三氯卡班减少软骨组织以缩小关节内间隙。
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Triclocarban stimulates DNMT1 to suppress collagen expression.
三氯卡班刺激 DNMT1 抑制胶原蛋白表达。
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The articular toxicity of triclocarban is conserved between zebrafish and human.
三氯卡班的关节毒性在斑马鱼和人类之间是保守的。

Abstract 抽象

Triclocarban (TCC) is a widely used environmental endocrine-disrupting chemical (EDC). Articular injury of EDCs has been reported; however, whether and how TCCs damage the joint have not yet been determined. Herein, we revealed that exposure to TCC caused osteoarthritis (OA) within the zebrafish anal fin. Mechanistically, TCC stimulates the expression of DNMT1 and initiates DNA hypermethylation of the type II collagen coding gene, which further suppresses the expression of type II collagen and other extracellular matrices. This further results in decreased cartilage tissue and narrowing of the intraarticular space, which is typical of the pathogenesis of OA. The regulation of OA occurrence by TCC is conserved between zebrafish cartilage tissue and human chondrocytes. Our findings clarified the hazard and potential mechanisms of TCC towards articular health and highlighted DNMT1 as a potential therapeutic target for OA caused by TCC.
三氯卡班（TCC）是一种广泛使用的环境内分泌干扰化学物质（EDC）。EDCs的关节损伤已有报道;然而，TCCs是否以及如何损害关节尚未确定。在此，我们发现暴露于TCC会导致斑马鱼臀鳍内的骨关节炎（OA）。从机制上讲，TCC刺激DNMT1的表达，启动II型胶原编码基因的DNA超甲基化，进一步抑制II型胶原和其他细胞外基质的表达。这进一步导致软骨组织减少和关节内间隙变窄，这是 OA 发病机制的典型特征。TCC对OA发生的调节在斑马鱼软骨组织和人软骨细胞之间是保守的。我们的研究结果阐明了TCC对关节健康的危害和潜在机制，并强调了DNMT1是TCC引起的OA的潜在治疗靶点。

Graphical Abstract 图形摘要

Keywords 关键字

Triclocarban

Osteoarthritis

Cartilage

Collagen

DNA methylation

三氯卡班骨关节炎软骨胶原DNA甲基化

1. Introduction 1. 引言

Triclocarban (TCC) is a highly effective and broad-spectrum antimicrobial agent that has been successfully used in personal care products, medical supplies, clothing, and even production of products for newborns [1]. Unfortunately, it is an environmental endocrine-disrupting chemical (EDC), and EDCs can disrupt the function of endocrine organs and thus threaten public health [2]. TCC and its intermediates in wastewater effluent, surface water, biosolids, sediment, and soil have been extensively reported [3], [4]. Moreover, recent investigations have demonstrated that the half-life of TCC is over 165 days under aerobic conditions [5]. The slow biodegradation of TCC leads to its efficient bioaccumulation and potential human exposure through food, water, and even dust [6]. As a result, TCC is frequently detected in body fluids, including blood, urine, and seminal plasma, thus posing a potential hazard to human health [7], [8].
三氯卡班（TCC）是一种高效、广谱的抗菌剂，已成功用于个人护理产品、医疗用品、服装，甚至新生儿产品的生产[1]。不幸的是，它是一种环境内分泌干扰化学物质（EDC），EDCs会破坏内分泌器官的功能，从而威胁公众健康[2]。TCC及其在废水、地表水、生物固体、沉积物和土壤中的中间体已被广泛报道[3]，[4]。此外，最近的研究表明，在有氧条件下，TCC的半衰期超过165天[5]。TCC的缓慢生物降解导致其有效的生物蓄积，并可能通过食物、水甚至灰尘暴露于人类[6]。因此，TCC经常在体液中检测到，包括血液、尿液和精浆，从而对人类健康构成潜在危害[7]，[8]。

The consequence of TCC exposure has been intensively investigated in different animal models and even human beings [2], [9]. Intensive studies have demonstrated that TCC and its analogues can also trigger metabolic disorders, including glucose homeostasis, lipid synthesis, lipid degradation, and transportation [10], [11], [12], [13]. In addition, studies have also indicated that exposure to TCC or its analogues might lead to teratogenesis during embryonic development [14], [15]. However, the primary target of TCC and its analogues is the endocrine system, similar to most EDCs [16], [17]. Investigations demonstrated that exposure to TCC could result in steroidal augmentation, masculinization, decreased fecundity and sex organ weights, and steroid-induced gene expression [18], [19].
TCC暴露的后果已经在不同的动物模型甚至人类中进行了深入研究[2]，[9]。大量研究表明，TCC及其类似物也可以引发代谢紊乱，包括葡萄糖稳态、脂质合成、脂质降解和运输[10]、[11]、[12]、[13]。此外，研究还表明，暴露于TCC或其类似物可能导致胚胎发育过程中的致畸[14]，[15]。然而，TCC及其类似物的主要靶点是内分泌系统，与大多数EDC相似[16]，[17]。研究表明，暴露于TCC可导致类固醇增强、男性化、生育力和性器官重量下降以及类固醇诱导的基因表达[18]，[19]。

It is well demonstrated that homeostasis of the reproductive endocrine system can maintain the health of the skeletal system [20]. For instance, osteoblasts and osteoclasts express androgen and oestrogen receptors; thus, androgen and oestrogen levels affect the number, activity, and apoptosis of these two kinds of cells [21], [22]. Although it has been known for some time that sex hormones affect the reaction of cartilage, little is known regarding how they influence cartilage homeostasis [18]. Recently, an epidemiological study investigated the impact of exposure to triclosan, an analogue of TCC, on the skeletal system. The results demonstrated that urinary triclosan concentration is positively associated with the prevalence of osteoporosis [23]. However, we still have limited knowledge about the impact of TCC on cartilage tissue.
生殖内分泌系统的稳态可以维持骨骼系统的健康[20]。例如，成骨细胞和破骨细胞表达雄激素和雌激素受体;因此，雄激素和雌激素水平会影响这两种细胞的数量、活性和凋亡[21]，[22]。尽管人们已经知道性激素会影响软骨的反应，但对它们如何影响软骨稳态知之甚少[18]。最近，一项流行病学研究调查了接触三氯生（TCC的类似物）对骨骼系统的影响。结果表明，尿三氯生浓度与骨质疏松症的患病率呈正相关[23]。然而，我们对TCC对软骨组织的影响仍然知之甚少。

Cartilage tissue facilitates smooth gliding contact between adjacent joint elements and maintains the health of the joint [24]. Cartilage degradation is a critical pathology of osteoarthritis (OA), and narrowing of the intraarticular space is a symptom of an advanced stage of OA [25]. As a degenerative disease, the significance of OA is increasing in parallel with life expectancy [26]. Investigation of whether and how environmental risk factors affect OA is of considerable socioeconomic and health care interest [27]. Herein, we showed that TCC reduces chondrogenic activity and triggers OA by studying zebrafish and human chondrocytes. Mechanistic studies indicate that TCC leads to the elevation of DNMT1, which maintains the hypermethylation of COL2A and decreases collagen formation. Our studies clarified the hazard and mechanism of TCC towards articular health and highlighted a potential therapeutic target for OA caused by TCC.
软骨组织促进相邻关节元件之间的平滑滑动接触，并维持关节的健康[24]。软骨退化是骨关节炎（osteoarthritis， OA）的关键病理学，关节内间隙变窄是骨关节炎晚期的症状[25]。作为一种退行性疾病，OA的重要性与预期寿命同步增加[26]。研究环境危险因素是否以及如何影响OA具有相当大的社会经济和医疗保健意义[27]。在此，我们通过研究斑马鱼和人类软骨细胞表明，TCC会降低软骨生成活性并触发OA。机制研究表明，TCC 导致 DNMT1 升高，从而维持 COL2A 的高甲基化并减少胶原蛋白的形成。我们的研究阐明了TCC对关节健康的危害和机制，并强调了TCC引起的OA的潜在治疗靶点。

2. Methods 2. 方法

2.1. Zebrafish line and zebrafish maintenance
2.1. 斑马鱼线和斑马鱼维护

The Casper zebrafish line was obtained from the China Zebrafish Resource Center (Wuhan, China). The zebrafish were maintained at 28.5 ± 0.5 °C with a pH of approximately 7.4 and a conductivity of approximately 550 μS/cm. Zebrafish exposed to TCC or DMSO were placed in individual tanks with self-circulating water, and the water was replaced daily. All experiments conducted in the current study were approved by the Research Ethics Committee of Jinan University.
Casper斑马鱼品系是从中国斑马鱼资源中心（中国武汉）获得的。斑马鱼保持在28.5±0.5°C，pH值约为7.4，电导率约为550μS/cm。将暴露于TCC或DMSO的斑马鱼放入装有自循环水的单独水箱中，并每天更换水。本研究进行的所有实验均已获得暨南大学研究伦理委员会的批准。

2.2. TCC quantification in synovial fluid of OA patients
2.2. OA患者滑液中的TCC定量

Chemicals and materials: Triclocarban was purchased from Beijing Solarbio Science and Technology Co., Ltd (Beijing, China). Clozapine was purchased from National Institutes for Food and Drug Control (Beijing, China). HPLC-grade acetonitrile, methanol and isopropanol were purchased from TEDIA (USA). Water was prepared using Elga Purelab Felx 3 water purification system (ELGA, High Wycombe, UK). All other chemicals and solvents were of the analytical grade available. Joint fluid was donated by the first Affiliated Hospital of Jinan University.
化学品和材料：三氯卡班购自北京索罗生物科技有限公司（中国北京）。氯氮平购自国家食品药品检定研究所（中国北京）。HPLC级乙腈、甲醇和异丙醇购自TEDIA（美国）。使用Elga Purelab Felx 3净水系统（ELGA，海威科姆，英国）制备水。所有其他化学品和溶剂均为可用分析级。关节液由暨南大学第一附属医院捐赠。

Patient sample preparation: To a 50 μL of joint fluid sample, 20 μL of the IS solution and 250 μL of acetonitrile were added. The samples were vortexed for 30 s and centrifuged for 10 min at 12,000 g. A 100 μL of the supernatant was transferred to an LC vial and diluted with 100 μL of water-acetonitrile (80/20: v/v). A 5 μL of the diluted supernatant was injected and analyzed by LC-MS/MS. All prepared samples were kept in an autosampler at 4 ℃ before injection.
患者样本制备：向 50 μL 关节液样本中加入 20 μL IS 溶液和 250 μL 乙腈。将样品涡旋30秒，并以12,000g离心10分钟。将 100 μL 上清液转移到 LC 小瓶中，并用 100 μL 水乙腈（80/20： v/v）稀释。进样 5 μL 稀释上清液，并通过 LC-MS/MS 进行分析。所有制备的样品在进样前保持在4°C的自动进样器中。

LC-MS/MS: LC-20 CE HPLC system (Shimadzu, Kyoto, Japan) was comprising a binary pump, a thermostatted autosampler and a thermostatted column compartment. An Ultimate XB-C18 column (5 µm, 50 ×2.1 mm, Welch, China) maintained at 40 ℃ was used for separation. A gradient program was used with the mobile phase, combining solvent A (methanol: acetonitrile: isopropanol 7:1.5:1.5, 0.2 % formic acid) and solvent B (10 mM ammonium formate in water) at a flow rate of 0.4 mL/min and with a total run time of 3.5 min as follows: 20 % A (0–0.2 min), 20–88 % A (0.21–1.3 min), 88–98 % A (1.31–1.8 min), 98 % A (1.81–2.4 min), 98–20 % A (2.41–2.5 min), 20 % A (3.51–3.5 min).
LC-MS/MS：LC-20 CE HPLC系统（日本京都岛津）由一个二元泵、一个恒温自动进样器和一个恒温柱温箱组成。使用保持在40°C的Ultimate XB-C18色谱柱（5μm，50×2.1mm，Welch，China）进行分离。流动相采用梯度程序，将溶剂A（甲醇：乙腈：异丙醇7：1.5：1.5,0.2%甲酸）和溶剂B（10mM甲酸铵溶液）以0.4 mL/min的流速和3.5 min的总运行时间组合在一起，如下所示：20%A（0-0.2分钟），20-88%A（0.21-1.3分钟）， 88-98 % A（1.31-1.8 分钟）、98% A（1.81-2.4 分钟）、98-20 % A（2.41-2.5 分钟）、20% A（3.51-3.5 分钟）。

The HPLC system was connected to a QTRAP 5500 mass spectrometer (AB Sciex, Singapore) equipped with an electrospray ionization (ESI) source. The Turbo-Ion-Spray interface was operated in positive-ion mode (MRM: triclocarban 314.1/161.1; clozapine 327.2/270.0) with nitrogen as the nebulizing, turbo spray, and curtain gas, with the optimum values set at 40, 40, and 35 psi, respectively. The turbo-gas temperature was set at 500 ℃, and the ESI needle voltage in positive-ion modes was adjusted to 4500 V. Instrument control and data processing was carried out by the Analyst 1.6.2 software. The retention times of triclocarban and clozapine was 2.72 and 2.26 min, respectively.
HPLC系统连接到配备电喷雾电离（ESI）源的QTRAP 5500质谱仪（AB Sciex，新加坡）。涡轮离子喷雾界面在正离子模式（MRM：三氯卡班 314.1/161.1;氯氮平 327.2/270.0）下运行，氮气作为雾化气体、涡轮喷雾和帘式气体，最佳值分别设置为 40、40 和 35 psi。涡轮气体温度设定为500°C，正离子模式下的ESI针电压调节为4500V。仪器控制和数据处理由Analyst 1.6.2软件进行。三氯卡班和氯氮平的保留时间分别为2.72和2.26 min。

Calibrators, quality control and internal standards preparation: Stock solutions of triclocarban and clozapine were prepared by dissolving these compounds in methanol at 0.2 and 1 mg/mL, respectively. The stock solution of triclocarban was further diluted with methanol to obtain working standard solutions at several concentrations. The calibration curve was obtained using six calibration standards, i.e., blank / joint fluid samples prepared by the addition of the working solutions to drug-free blank plasma/joint fluid sample, giving final concentrations of 0.5, 1, 2.5, 5, 8, 10 ng/mL for triclocarban. Calibration curves for triclocarban in human plasma/joint fluid were derived from their peak area ratios relative to that of internal standard (IS) using linear regression with 1/x² as a weighting factor. Quality control (QC) samples were prepared at three concentration levels (low, medium and high) for final concentrations of 1.5, and 7.5 ng/mL. The stock solution of clozapine was diluted to 200 ng/mL in methanol for routine use as a IS. All prepared plasma/joint fluid samples and stock solutions were stored at − 80 ℃.
校准品、质量控制和内标制备：通过将三氯卡班和氯氮平分别溶解在甲醇中以 0.2 mg/mL 和 1 mg/mL 溶解来制备这些储备溶液。用甲醇进一步稀释三氯卡班的储备溶液，得到几种浓度的工作标准溶液。使用六种校准标准获得校准曲线，即通过将工作溶液添加到无药物空白血浆/关节液样品中制备的空白/接头液样品，三氯卡班的终浓度为 0.5、1、2.5、5、8、10 ng/mL。人血浆/关节液中三氯卡班的校准曲线是根据其相对于内标（IS）的峰面积比得出的，采用以 1/x ² 为加权因子的线性回归。在三种浓度水平（低、中和高）下制备质量控制（QC）样品，最终浓度分别为 1.5 和 7.5 ng/mL。将氯氮平的储备溶液在甲醇中稀释至200 ng/mL，作为IS常规使用。所有制备的血浆/关节液样品和储备溶液均储存在-80°C。

Calibration curves were extracted in duplicate by adding the following volumes to 50 μL of joint fluid: 50 μL of the standard, 20 μL of the IS solution and 200 μL of acetonitrile. The standards were vortexed for 30 s and centrifuged for 10 min at 12,000 g. An aliquot of 100 μL of the supernatant was transferred to LC vial and diluted with 100 μL of water-acetonitrile (80/20: v/v). A 5 μL aliquot of the diluted supernatant was injected and analyzed by LC-MS/MS. The calibration curve for triclocarban was linear over the concentration ranges, with determination coefficients (r²) higher than 0.99.
通过向 50 μL 接头液中加入以下体积来提取校准曲线：50 μL 标准品、20 μL IS 溶液和 200 μL 乙腈。将标准品涡旋30秒，并以12,000g离心10分钟。将 100 μL 上清液的等分试样转移到 LC 小瓶中，并用 100 μL 水-乙腈（80/20： v/v）稀释。进样5 μL稀释上清液的等分试样，并通过LC-MS/MS进行分析。三氯卡班的校准曲线在浓度范围内呈线性，决定系数（r ² ）高于0.99。

Cells and cell culture: Human primary chondrocytes were purchased from ICell Bioscience Incorporated (Shanghai, China). Chondrocytes from six different healthy donors were employed in this study. All primary chondrocytes were characterized by col2a-positive immunofluorescent staining (Fig. S7). The cells were maintained in the standard medium provided by the company. Subculture was performed when the cells grew to 85 % confluence at a 1:2 ratio. The cells were incubated at 37 °C with 5 % CO₂.
细胞和细胞培养：人原代软骨细胞购自ICell Bioscience Incorporated（中国上海）。本研究使用了来自六个不同健康供体的软骨细胞。所有原代软骨细胞均通过col2a阳性免疫荧光染色进行表征（图S7）。细胞保持在公司提供的标准培养基中。当细胞以 1：2 的比例生长至 85% 汇合时进行传代培养。将细胞在37°C与5%CO一起孵育 ₂ 。

2.3. Zebrafish imaging 2.3. 斑马鱼成像

Imaging of the anaesthetized or stained zebrafish was performed under a stereomicroscope (Olympus SZX7, Ming Mei, China). Hyperaemia was quantified by a virtual ruler in the software of the imaging system. Briefly, the virtual ruler was first calibrated by a physical ruler, and the calibrated virtual ruler was employed to detect the length of the whole anal fin (L0) and the length of the hyperemic area (Lh) in a double-blind manner. The hyperaemia rate is presented as 100 % X (Lh/L0).
在体视显微镜下对麻醉或染色的斑马鱼进行成像（奥林巴斯SZX7，明美，中国）。充血在成像系统软件中通过虚拟标尺进行量化。简言之，首先用物理尺对虚拟尺子进行校准，并利用校准后的虚拟尺子以双盲方式检测整个臀鳍的长度（L0）和充血区的长度（Lh）。充血率表示为 100 % X （Lh/L0）。

2.4. Heartbeat rate 2.4. 心跳率

The treated zebrafish larva was placed on an agarose gel plate under a stereomicroscope (Olympus SZX7, Ming Mei, China), and the heartbeats were recorded over the course of a minute in a double-blind manner. The heartbeat rate was indicated as times/min.
将处理过的斑马鱼幼虫置于立体显微镜下的琼脂糖凝胶板上（奥林巴斯SZX7，Ming Mei，China），并以双盲方式记录一分钟的心跳。心跳率以次/分钟表示。

2.5. Alcian blue and alizarin red double staining
2.5. 阿尔新蓝和茜素红双重染色

The treated zebrafish were dissected, fixed with paraformaldehyde, and maintained in absolute ethanol. Staining was performed using 1 % Alcian Blue dissolved in 20 % glacial acetic acid and 80 % EtOH and 0.01 % alizarin red dissolved in 1 % KOH according to the standard protocol described in reference [28]. The stained zebrafish were placed on an agarose gel plate under a stereomicroscope (Olympus SZX7, Ming Mei, China). The intraarticular space was quantified by a virtual ruler in the imaging system. Briefly, the virtual ruler was first calibrated by a physical ruler, and the calibrated virtual ruler was employed to detect the shortest distance between the bone of the joint, which was stained with alizarin red in a double blind manner. The intraarticular space is presented in μm.
将处理过的斑马鱼解剖，用多聚甲醛固定，并维持在无水乙醇中。根据参考文献中描述的标准方案，使用溶于20%冰醋酸中的1%Alcian Blue和溶于1%KOH的80%EtOH和0.01%茜素红进行染色[28]。将染色的斑马鱼放在立体显微镜下的琼脂糖凝胶板上（奥林巴斯SZX7，明美，中国）。关节内空间由成像系统中的虚拟尺子量化。简言之，首先用物理尺对虚拟尺子进行校准，并利用校准后的虚拟尺子检测关节骨之间的最短距离，以双盲方式用茜素红染色。关节内间隙以 μm 表示。

2.6. Metabolome analysis 2.6. 代谢组分析

Treated zebrafish were sacrificed in ice water, and the articular areas of the anal fin were dissected and subjected to metabolomics analysis. Metabolite extraction was primarily performed according to the following methods. In short, 25 mg tissues were weighed and extracted by directly adding 800 μL of precooled extraction reagent (methanol:acetonitrile:water (2:2:1, v/v/v)), internal standards mix 1 (IS1), and internal standards mix 2 (IS2) for quality control of sample preparation. After homogenization for 5 min using TissueLyser (JXFSTPRP, China), the samples were sonicated for 10 min and incubated at − 20 °C for 1 h. The samples were centrifuged for 15 min at 25,000 rpm at 4 °C, and the supernatant was then transferred for vacuum freeze drying. The metabolites were resuspended in 200 μL of 10 % methanol and sonicated for 10 min at 4 °C after centrifugation for 15 min at 25,000 rpm, and the supernatants were transferred to autosampler vials for LCMS analysis. A quality control (QC) sample was prepared by pooling the same volume of each sample to evaluate the reproducibility of the whole LCMS analysis. LCMS/MS was employed for untargeted metabolomics analysis. Data from both positive and negative ions were collected by a high-resolution mass spectrometer Q Exactive HF (Thermo Fisher Scientific, USA) to improve the coverage. After MS analysis, the data were processed in Compound Discoverer 3.1 (Thermo Fisher Scientific, USA) software. Metabolome bioinformatic analysis was performed using the metabolomics R package metaX developed by BGI (Shenzhen, China). The fold change and Student's t test were used to screen for differential metabolites.
将处理过的斑马鱼置于冰水中，解剖臀鳍的关节区域并进行代谢组学分析。代谢物提取主要按照以下方法进行。简言之，通过直接加入800 μL预冷提取试剂（甲醇：乙腈：水（2：2：1，v/v/v））、内标混合物1（IS1）和内标混合物2（IS2）来称取25 mg组织，用于样品制备的质量控制。使用TissueLyser（JXFSTPRP，China）匀浆5分钟后，将样品超声处理10分钟，并在-20°C下孵育1小时。将样品在4°C下以25,000rpm离心15分钟，然后转移上清液进行真空冷冻干燥。将代谢物重悬于200μL10%甲醇中，并在25,000rpm离心15分钟后在4°C超声处理10分钟，并将上清液转移到自动进样器样品瓶中进行LC MS分析。通过合并每个样品的相同体积来制备质量控制（QC）样品，以评估整个 LC MS 分析的重现性。LC MS/MS用于非靶向代谢组学分析。高分辨率质谱仪Q Exactive HF（美国赛默飞世尔科技）收集了来自正离子和负离子的数据，以提高覆盖率。MS分析后，数据在Compound Discoverer 3.1（Thermo Fisher Scientific，USA）软件中处理。使用华大基因（中国深圳）开发的代谢组学R包metaX进行代谢组生物信息学分析。采用倍数变化和Student's t检验筛选差异代谢物。

2.7. Transcriptome analysis
2.7. 转录组分析

Treated zebrafish were sacrificed in ice water, and the articular areas of the anal fin were dissected for mRNA sequencing. Total RNA was extracted using the RNeasy Micro Kit (Cat# 74004, Qiagen). Quantification and qualification were performed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). The library was constructed following the instructions of the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina® before being subjected to an Illumina NovaSeq6000 sequencer. The expression abundance is presented as fragments per kilobase million (FPKM). Differential expression analysis was conducted using DESeq2 software [29]. Genes with a parameter of false discovery rate (FDR)≤ 0.05 and absolute fold change≥ 2 were filtered as differentially expressed genes (DEGs).
将处理过的斑马鱼置于冰水中，并解剖臀鳍的关节区域进行mRNA测序。使用 RNeasy Micro Kit（货号# 74004，Qiagen）提取总 RNA。使用 Agilent Bioanalyzer 2100（Agilent Technologies，Santa Clara，CA，US）进行定量和鉴定。在对Illumina NovaSeq6000测序仪进行测序之前，按照用于Illumina®的VAHTS Universal V6 RNA-seq文库制备试剂盒的说明构建了该文库。表达丰度表示为每千碱基百万片段数（FPKM）。使用DESeq2软件进行差异表达分析[29]。将错误发现率（FDR）参数为0.05≤绝对倍数变化≥2的基因过滤为差异表达基因（DEGs）。

2.8. GO analysis 2.8. GO分析

DEGs were mapped to the Gene Ontology (GO) database using the Omicshare online software (https://www.omicshare.com/tools/). A hypergeometric test defined significantly enriched GO terms among the DEGs.
使用 Omicshare 在线软件（https://www.omicshare.com/tools/）将 DEG 映射到基因本体（GO）数据库。超几何测试定义了 DEG 中显着丰富的 GO 项。

2.9. KEGG analysis 2.9. KEGG分析

DEGs were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using Omicshare online software (https://www.omicshare.com/tools/). A hypergeometric test defined significantly enriched KEGG pathways among the DEGs.
使用Omicshare在线软件（https://www.omicshare.com/tools/）将DEG映射到京都基因和基因组百科全书（KEGG）数据库。超几何测试定义了 DEG 中显着富集的 KEGG 通路。

2.10. GSEA 2.10. GSEA公司

All genes with FPKM changes were analysed by Gene Set Enrichment Analysis (GSEA) using Omicshare online software (https://www.omicshare.com/tools/). A hypergeometric test defined significantly enriched gene sets among the DEGs.
使用Omicshare在线软件（https://www.omicshare.com/tools/）通过基因集富集分析（GSEA）分析所有具有FPKM变化的基因。超几何测试定义了 DEG 中显着富集的基因集。

2.11. Sirius red staining
2.11. 天狼星红染色

Chondrocytes were seeded in 48-well plates at 105 cells/mL. Different concentrations of TCC and its control solvent, DMSO, were added to the cells for 72 h. The treated cells were then fixed with paraformaldehyde and washed with PBS. Picrosirius Red Solution (Abcam, US) was applied for 60 min. The stained cells were then rinsed with an acetic acid solution (0.5 %). The cells were observed and imaged under an inverted microscope (Olympus ix70, Japan). The Sirius red staining of the cells was then quantified according to the gradient densitometry using ImageJ software in a double-blind manner.
将软骨细胞接种在 48 孔板中，浓度为 105 个细胞/mL。将不同浓度的TCC及其对照溶剂DMSO加入细胞中72小时。然后用多聚甲醛固定处理过的细胞，并用PBS洗涤。Picrosirius Red 溶液（Abcam，US）涂抹 60 分钟。然后用醋酸溶液（0.5%）冲洗染色的细胞。在倒置显微镜（Olympus ix70，日本）下观察和成像细胞。然后使用ImageJ软件以双盲方式根据梯度密度测定法对细胞的天狼星红染色进行定量。

2.12. Real-time quantified PCR
2.12. 实时定量PCR

Total RNA was extracted using a RNeasy Micro Kit (Cat# 74004, Qiagen). Reverse transcription was conducted by following the standard protocol of the PrimerScript RT kit (Takara, Japan). The qPCR was performed with a thermal cycling condition of 95 °C (denature) for 15 s and 60 °C (anneal and elongation) using a qPCR mix (MCE, New Jersey, US) on a Step One Real-Time system (ABI, USA). The expression of beta actin (ACTB) was used as a housekeeping gene, and the gene expression is presented as the fold change compared to the control. Three biological repeats were performed for each treatment group.
使用 RNeasy Micro Kit（货号# 74004，Qiagen）提取总 RNA。按照PrimerScript RT试剂盒（日本Takara）的标准方案进行逆转录。在Step One实时系统（ABI，USA）上使用qPCR混合物（MCE，New Jersey，US）在95°C（变性）和60°C（退火和伸长率）的热循环条件下进行qPCR。β肌动蛋白（ACTB）的表达被用作管家基因，基因表达表现为与对照组相比的倍数变化。对每个治疗组进行三次生物学重复。

PCR primers: PCR引物：

hCOL2A1-F: 5’- CCTGGCAAAGATGGTGAGACAG.
hCOL2A1-F：5'-CCTGGCAAAGATGGTGAGACAG。

hCOL2A1-R: 5’-CCTGGTTTTCCACCTTCACCTG.
hCOL2A1-R：5'-CCTGGTTTTCCACCTTCACCTG。

hCOL9A1-F: 5’- GGCAGTAGAGGAGAATTAGGACC.
hCOL9A1-F：5'-GGCAGTAGAGGAGAATTAGGACC。

hCOL9A1-R: 5’- GTTCACCGACTACACCCCTG.
hCOL9A1-R：5'-GTTCACCGACTACACCCCTG。

hCOL9A3-F: 5’- GTGGATGGTCTGACTGGACG.
hCOL9A3-F：5'-GTGGATGGTCTGACTGGACG。

hCOL9A3-R: 5’- GGGCAGATACTTGGGCACTG.
hCOL9A3-R：5'-GGGCAGATACTTGGGCACTG。

hCOL5A2-F: 5’- CCTGGCAAAGATGGTGAGACAG.
hCOL5A2-F：5'-CCTGGCAAAGATGGTGAGACAG。

hCOL5A2-R: 5’-CCTGGTTTTCCACCTTCACCTG.
hCOL5A2-R：5'-CCTGGTTTTCCACCTTCACCTG。

hDNMT1-F: 5’- CGAGGAAGTAGAAGCGGTTGG.
hDNMT1-F：5'-CGAGGAAGTAGAAGCGGTTGG。

hDNMT1-R: 5’- AAGGAGCCCGTGGATGAGG.
hDNMT1-R：5'- AAGGAGCCCGTGGATGAGG。

hATCB-F: 5’-CACCATTGGCAATGAGCGGTTC.
hATCB-F：5'-CACCATTGGCAATGAGCGGTTC。

hATCB-R: 5’-AGGTCTTTGCGGATGTCCACGT.
hATCB-R：5'-AGGTCTTTGCGGATGTCCACGT。

2.13. Methylation-specific PCR
2.13. 甲基化特异性PCR

Zebrafish exposure to TCC was executed in ice water, and the articular areas of the anal fin were dissected and treated with bisulfite. The TCC-exposed chondrocytes were also treated with bisulfite. DNA samples from zebrafish articular areas and chondrocytes were then extracted and purified following the instructions of the Methylation Specific PCR Kit (EM101, Tiangen, China). The methylation-specific and unmethylation-specific primers were designed through the website http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi. The methylation-specific PCR was performed under the same conditions as the real-time quantified PCR section.
斑马鱼暴露于TCC在冰水中进行，解剖臀鳍的关节区域并用亚硫酸氢盐处理。暴露于TCC的软骨细胞也用亚硫酸氢盐处理。然后按照甲基化特异性PCR试剂盒（EM101，Tiangen，China）的说明提取和纯化斑马鱼关节区域和软骨细胞的DNA样本。甲基化特异性和非甲基化特异性引物是通过网站 http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi 设计的。甲基化特异性PCR在与实时定量PCR切片相同的条件下进行。

Methylation-specific primers:
甲基化特异性引物：

hCOL2A1-MF: 5’- TGGCCAGGGCCGCAGCAGTGACCAACC.
hCOL2A1-MF：5'-TGGCCAGGGCCGCAGCAGTGACCAACC。

hCOL2A1-MR: 5’- GACTGAAAGAGCCACAGAGGACGGAG.
hCOL2A1-MR：5'-GACTGAAAGAGCCACAGAGGACGGAG。

hCOL2A1-UF: 5’-TTAGGGTCGTAGTAGTGATTAATCGT.
hCOL2A1-UF：5'-TTAGGGTCGTAGTAGTGATTAATCGT。

hCOL2A1-UR: 5’-AACTAAAAAAACCACAAAAAACGAAA.
hCOL2A1-UR：5'-AACTAAAAAAACCACAAAAAACGAAA。

hATCB-F: 5‘-AGCGATGCGTTCGAGCATCGCUTAGGGAGTATATAGGTTGGGGAAGTT.
hATCB-F：5'-AGCGATGCGTTCGAGCATCGCUTAGGGAGTATAGGTTGGGGAAGTT。

hATCB-R: 5‘-AACACACAATAACAAACACAAATTCAC.
hATCB-R：5'-AACACACAACAAACACACAAATTCAC。

2.14. Western blotting 2.14. 蛋白质印迹

Western blotting was performed to measure the protein level of type II collagen. Briefly, the chondrocytes were seeded in 6 cm cell plates and treated with different concentrations of TCC (0.03–3 μM) for 72 h. The treated cells were then harvested using cell scrapers and lysed using RIPA buffer (HY-K1001, MCE, US). The cell lysate was then subjected to noncontact sonication for 10 min followed by centrifugation at 10000XG for 10 min at 4 °C. The supernatant was collected, and BCA was employed to quantify the total protein, followed by boiling with loading buffer (S3014, Merk, US). The same protein samples were loaded for SDSPAGE. After electrophoresis, the proteins were transferred to a PVDF membrane and blocked with 5 % nonfat milk at room temperature. The blocked membrane was then incubated with COL2A1 primary antibody (ab239007, Abcam, US) at 4 °C overnight. The membrane was washed three times with TBST before incubation with HRP-linked anti-rabbit antibody (ab205718, Abcam, US). Another three rounds of TBST washing were performed before developing the protein bands with ECL reagents (WBULS0100, Merck, US). GAPDH (ab9485, Abcam, US) was used as a loading control.
进行蛋白质印迹以测量II型胶原蛋白水平。简而言之，将软骨细胞接种在6cm细胞板中，并用不同浓度的TCC（0.03-3μM）处理72小时。然后使用细胞刮刀收获处理过的细胞，并使用RIPA缓冲液（HY-K1001，MCE，US）裂解。然后对细胞裂解物进行非接触式超声处理10分钟，然后在4°C下以10000XG离心10分钟。收集上清液，使用BCA定量总蛋白，然后用上样缓冲液（S3014，Merk，US）煮沸。为SDS PAGE加载相同的蛋白质样品。电泳后，将蛋白质转移到PVDF膜上，并在室温下用5%脱脂牛奶封闭。然后将封闭的膜与COL2A1一抗（ab239007，Abcam，US）在4°C下孵育过夜。用 TBST 洗涤膜 3 次，然后与 HRP 连接的抗兔抗体孵育（ab205718， Abcam， US）。在用 ECL 试剂（WBULS0100，Merck，US）开发蛋白条带之前，再进行三轮 TBST 洗涤。GAPDH （ab9485， Abcam， US）用作装载控制。

2.15. Statistical analysis
2.15. 统计分析

Statistical data analysis was conducted using GraphPad Prism 9.0 on Macintosh OS X, and all data are expressed as the mean ± SD. One-way ANOVA and multiway ANOVA were used to perform a significant analysis of multiple groups, and significant differences were considered when the p value was < 0.05.
在Macintosh OS X上使用GraphPad Prism 9.0进行统计数据分析，所有数据均以平均值±标准差表示，采用单因素方差分析和多因素方差分析对多组进行显著分析，当p值<0.05时考虑显著差异。

3. Results 3. 结果

3.1. TCC triggers osteoarthritis in the zebrafish anal fin
3.1. TCC引发斑马鱼臀鳍骨关节炎

As the first step, we quantified the OA in the joint fluid from articulatio genus of 15 OA patients and 10 non-OA donors. Results demonstrated that the average TCC concentration in OA patients reaches 2 ng/mL, which is significantly higher than that in non-OA donors (0.15 ng/mL) (Fig. S1). The acute toxicity of TCC was then evaluated in zebrafish larvae. Although the morphology of zebrafish larvae was healthy overall, the decrease in the larval survival rate became significant when the TCC concentration was increased to 10 μM (Fig. S2A-B). The heartbeat rate of zebrafish larvae decreased after exposure to 1 μM and 3 μM TCC for 72 h, although they the zebrafish were still alive at those concentrations (Fig. S2C). The adult zebrafish demonstrated more resistance to TCC, as indicated by the unchanged stander length and body weight after long-term exposure to TCC for three weeks (Fig. S2D-E). However, we found that chronic exposure to TCC resulted in hyperaemia around the joints of the anal fin in adult zebrafish in a time- and dose-dependent manner (Fig. 1 and Fig. S3). These results suggested that TCC may injure the joints in adult zebrafish.
作为第一步，我们量化了 15 名 OA 患者和 10 名非 OA 供体的关节液中的 OA。结果表明，OA患者的平均TCC浓度达到2 ng/mL，显著高于非OA供体（0.15 ng/mL）（图S1）。然后评估TCC在斑马鱼幼虫中的急性毒性。虽然斑马鱼幼虫的形态总体上是健康的，但当TCC浓度增加到10μM时，幼虫存活率的下降变得明显（图S2A-B）。斑马鱼幼虫在暴露于1μM和3μM TCC72小时后心跳率降低，尽管斑马鱼在这些浓度下仍然存活（图S2C）。成年斑马鱼对TCC表现出更强的抵抗力，如长期暴露于TCC三周后站立长度和体重不变所表明（图S2D-E）。然而，我们发现长期暴露于TCC导致成年斑马鱼臀鳍关节周围充血，其时间和剂量依赖性（图1和图S3）。这些结果表明，TCC可能会损伤成年斑马鱼的关节。

To further investigate the impact of TCC on the articular area around the anal fin, Alcian blue and alizarin red double staining was performed, and the results indicated that the bone and cartilage tissue were well stated in zebrafish exposed to TCC for three weeks (Fig. 2 A). Interestingly, we found that the intraarticular space narrowed after exposure to TCC (Fig. 2B). We further quantified the intraarticular space in the anal fin. The healthy zebrafish demonstrated an average intraarticular distance of approximately 16 µm. In contrast, the distance diminished to only one-half after exposure to TCC (Fig. 2 C), suggesting that TCC might cause OA in zebrafish anal fins.
为进一步研究TCC对臀鳍周围关节区的影响，采用阿尔新蓝和茜素红双重染色，结果表明，暴露于TCC3周的斑马鱼的骨骼和软骨组织均有良好的表现（图2A）。有趣的是，我们发现暴露于TCC后关节内空间变窄（图2B）。我们进一步量化了臀鳍的关节内空间。健康的斑马鱼的平均关节内距离约为 16 μm。相比之下，暴露于TCC后，距离减少到只有一半（图2 C），这表明TCC可能导致斑马鱼臀鳍OA。

3.2. TCC programs an osteoarthritis-prone metabolome
3.2. TCC对骨关节炎易感代谢组进行编程

To validate whether TCC triggered OA in zebrafish, the articular area of the anal fin was dissected and subjected to untargeted metabolomics and transcriptomics analysis (Fig. S4A). The metabolites in the articular area significantly changed after TCC exposure in both positive and negative ion modes, as indicated by tables (Tables S2 and S3), volcano diagrams (Fig. S4B and C), and heatmaps (Fig. 3A-B). By using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), the distribution of metabolites demonstrated good categorization between TCC and its solvent control group in both positive and negative ion modes (Fig. 3C-F). These differential metabolites were further analysed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). As indicated in Fig. 3 G, several pathways, such as the citrate cycle, fatty acid biosynthesis pathway, and Foxo1 signalling pathway, were significantly enriched in the joints of the anal fin of zebrafish exposed to TCC. These pathways are also significantly altered in clinical OA patients [30], [31], [32]. We further analysed the metabolites that were significantly changed after exposure to TCC. Table S1 demonstrates the intersection of between the increased and decreased metabolites in the joints exposed to TCC and the significantly changed metabolites in the joint fluids of OA patients according to the previous clinical studies. Most of these metabolites are highly consistent between TCC-exposed zebrafish and clinical OA patients. These results confirm that TCC triggers OA at the molecular level.
为了验证TCC是否触发了斑马鱼的OA，解剖了臀鳍的关节区域，并进行了非靶向代谢组学和转录组学分析（图S4A）。在TCC暴露后，关节区域的代谢物在正离子和负离子模式下都发生了显着变化，如表（表S2和S3）、火山图（图S4B和C）和热图（图3A-B）所示。通过使用主成分分析（PCA）和偏最小二乘判别分析（PLS-DA），代谢物的分布在正离子和负离子模式下都显示出TCC及其溶剂对照组之间的良好分类（图3C-F）。根据《京都基因和基因组百科全书》（KEGG）对这些差异代谢物进行了进一步分析。如图3 G所示，枸橼酸循环、脂肪酸生物合成途径和Foxo1信号通路等几种途径在暴露于TCC的斑马鱼臀鳍关节中显著富集。这些通路在临床OA患者中也发生显著改变[30]，[31]，[32]。我们进一步分析了暴露于TCC后发生显着变化的代谢物。表S1显示了根据先前的临床研究，暴露于TCC的关节中代谢物的增加和减少与OA患者关节液中代谢物显着变化之间的交集。这些代谢物中的大多数在暴露于 TCC 的斑马鱼和临床 OA 患者之间高度一致。这些结果证实了TCC在分子水平上触发OA。

3.3. TCC suppresses chondrogenesis and extracellular matrix gene expression

Transcriptomics analysis of the articular area of the anal fin was also performed, and the differentially expressed genes (DEGs) are presented in a volcano diagram (Fig. S5A) and heatmap (Fig. 4A). We further performed gene set enrichment analysis (GSEA) of the transcriptome results, and the top 5 upregulated and downregulated gene sets are indicated in Fig. S5B. Interestingly, we found that the extracellular matrix structural constituent and the collagen trimer gene sets, which are critical for cartilage tissue, are the two most enriched gene sets in the downregulated list. We then analysed the related gene sets and found that several extracellular matrix (ECM)- and collagen-related gene sets were overall downregulated after TCC exposure, as presented in Fig. 4B.

Collagen is a crucial ECM component of cartilage tissue. Therefore, the collagen trimer set genes are indicated in a lattice diagram based on the GSEA results (Fig. 4C). Almost all collagen coding genes were inhibited by TCC exposure. Type II collagen is the primary ECM in cartilage tissue. Moreover, the reduced cartilage tissue-related ECM genes, including col2a1a, and elevated inflammation-related genes are indicated in Fig. 4D. The col2a1a gene, the major gene that codes Type II collagen, is highlighted in the lattice diagram, and it also demonstrated a marked decline after TCC exposure. The progesterone−mediated oocyte maturation-related genes were enriched among the upregulated pathways. We also found that TCC might bind to the progesterone receptor (PGR) at its ligand-binding domain (Fig. S6). These results suggested that TCC triggers a decrease in cartilage tissue at the transcriptional level.

Primary human chondrocytes for six non-OA donors were introduced to investigate whether TCC suppresses the expression of the type II collagen coding gene conserved in humans. The primary human chondrocytes were characterized by immunofluorescence for type II collagen (Fig. S7). As shown in Fig. 5 A and B, TCC exposure caused a dose-dependent collagen decline, as indicated by the gradually weakened Sirius red staining. Expression of the human COL2A gene demonstrated a dose-dependent decrease at both the mRNA and protein levels after exposure to TCC (Fig. 5 C and D). These results suggested that the collagen formation inhibitory effect of TCC is conserved between zebrafish and human beings.

3.4. TCC stimulates DNMT1 expression and methylation of the type II collagen coding gene

To further investigate the potential upstream regulator of type II collagen coding gene expression in response to TCC, we predicted the potential transcription factors of COL2A1; however, none of the possible transcription factors changed after TCC exposure (Fig. S3B). We then reanalyzed the transcriptome results and found that the DNA methylation genes had significantly different gene ontology (GO) analysis results (Fig. 5 F). Moreover, the GSEA lattice diagram also demonstrated that DNA metabolism and negative regulation of gene expression subsets were significantly elevated after TCC exposure. Interestingly, DNA methyltransferase 1 (DNMT1), highlighted in Fig. 5 G and H, belongs to both gene subsets. We also quantified the expression in human chondrocytes, and the results demonstrated that the DNMT1 transcript gradually increased when the TCC concentration increased (Fig. 5E), indicating that DNMT1 expression stimulated by TCC is conserved between zebrafish and human beings.

Usually, DNMT1 maintains DNA methylation and thus suppresses the expression of methylated genes [30]. We then scanned human COL2A1 (Fig. 6 A) and zebrafish col2a1a (Fig. 6B) genes. There are GC-rich sequences in both of their 5’ UTR regions. Bisulfite-based qPCR was employed to evaluate the methylation of the GC-rich sequence of human COL2A1 and zebrafish col2a1a genes. As indicated in Fig. 6 C, the solvent control demonstrated a higher unmethylated level in the GC-rich sequence of the COL2A1 gene than that in the TCC-exposed chondrocytes using unmethylated primers (UP). When using methylated primers (MPs), TCC-exposed chondrocytes demonstrated higher methylation levels in the GC-rich sequence of the COL2A1 gene than solvent control cells, indicating that TCC stimulated DNA methylation in the GC-rich sequence of the COL2A1 gene in human chondrocytes. The methylation of the zebrafish col2a1a gene was also quantified in the articular tissue of zebrafish anal fins, and the results were similar to those of human chondrocytes (Fig. 6D). These results collectively demonstrated that the methylation of the GC-rich sequence in COL2A1/col2a1a genes stimulated by TCC is conserved between zebrafish and human beings.

3.5. TCC suppresses COL2A1 via DNMT1-dependent DNA methylation

To confirm the role of DNMT1 in the suppression of type II collagen expression by TCC, two DNMT1 inhibitors, DC_517 and CM-272, were introduced to pretreat human chondrocytes before exposure to TCC. As shown in Fig. 6E, the methylation of the COL2A1 gene was also quantified, and the bisulfite-based qPCR results demonstrated that both DC_517 and CM-272 abolished the methylation of COL2A1 induced by TCC. Moreover, TCC significantly suppressed COL2A1 expression in a dose-dependent manner, while the inhibitory effect of TCC became insignificant when the chondrocytes were pretreated with either DC_517 or CM-272 (Fig. 6 F). Quantification was also performed for COL9A1, COL9A3 and COL5A2, which encode other core proteins of type II collagen. Fig. S8 demonstrated that TCC decreased the expression of COL9A1, COL9A3 and COL5A2, which is consistent with the expression of zebrafish col9a1a, col9a3 and col5a2b (Fig. 4D). Two DNMT1 inhibitors, DC-517 and CMC-272, significantly reversed the decreased expression COL9A1, COL9A3 and COL5A2 induced by TCC, indicating the TCC supressed the expression of COL9A1, COL9A3 and COL5A2 in a DNMT1-dependent manner. Sirius red staining of human chondrocytes demonstrated that inhibition of DNMT1 by either DC_517 or CM-272 abolished the suppressive effect of TCC on collagen formation (Fig. 6 G). These results indicated that TCC suppresses COL2A1 via DNMT1-dependent DNA methylation.

4. Discussion

The correlation between EDCs and OA occurrence has been identified. However, there is still no direct evidence regarding whether and how TCC exposure contributes to OA. Herein, we demonstrated that TCC triggers OA occurrence in vitro and in vivo. The articular toxicity of TCC is conserved between zebrafish and human beings. Mechanistically, TCC stimulates DNMT1 expression and thus methylates and suppresses the type II collagen coding gene in cartilage. The intraarticular space narrowed because of the decline in collagen-based ECM in the cartilage tissue, followed by the occurrence of OA. Our results highlight TCC as a potential environmental threat to articular health.

Recently, the toxicities of environmental pollutants, especially EDCs, have attracted increasing attention. Studies have demonstrated that many EDCs can damage the skeletal system through endocrine disruption [23]. EDCs, for instance, phenols, parabens, and perfluorinated compounds, have been identified as environmental risks that contribute to the occurrence of OA [27]. TCC is a member of the EDC family; however, its articular toxicity and toxicology have not yet been defined [31]. This study is one of the leading investigations uncovering the articular toxicity of TCC in vitro and in vivo.

Mechanistically, bisphenol A and benzophenone-3 were highlighted as ligands with weak activities against oestrogen receptors [32]. They bound oestrogen receptors and decreased oestrogen receptor signalling in females but increased oestrogen receptor signalling in males [33]. This endocrine disruption can stimulate NF-κB signalling [34], which contributes to a proinflammatory microenvironment during the pathogenesis of OA. In our study, transcriptome analysis indicated that NF-κB signalling might also be involved in TCC-induced OA. Perfluorooctanoic acid and perfluorooctane sulfonate also damage the joints [35], [36]. These perfluorinated compounds triggered OA by suppressing parathyroid hormone two receptor expression [37]. The direct signalling responsible for the OA occurrence induced by these EDCs has yet to be defined. Our current study discovered that TCC triggers the expression of DNMT1, which further catalyses the methylation of the type II collagen coding gene and suppresses its expression in chondrocytes. We are one of the first to discover the articular toxicity of EDCs through the disruption of epigenetic modification. Our results indicate that DNMT1 might be a potential therapeutic target for OA caused by TCC.

Zebrafish is one of the most popular animal models for studying aqueous toxicity and toxicology [38]. The toxicities of environmental pollutants were intensively investigated using zebrafish. For instance, the toxicities of bisphenol A towards gonad development and lipid metabolism were demonstrated in zebrafish [39]. However, the impact of these chemicals on the joints has rarely been investigated. The OA phenotype was first identified a decade ago and has been further investigated in zebrafish [40]. Suspect mutations in osteoarthritis are also critical for joint shape during ontogeny in zebrafish. Genes, such as Chsy1 and col11a2, were markedly decreased during OA and caused OA in zebrafish [41], [42]. These results suggested that the regulatory OA occurrence might be conserved between zebrafish and human beings. In this study, we discovered that TCC, a EDC, triggers OA in zebrafish anal fins, and the mechanism is conserved in human chondrocytes. Our current study supports the idea that zebrafish is an appropriate animal model for investigating OA.

The epigenetic toxicity of EDCs has been reported. Although changes in the modification of histones induced by TCC have yet to be identified [43], TCC stimulated the hypermethylation of AhR and Esr1 in mouse neuronal cells [44], [45]. In zebrafish, TCC also triggers the hypermethylation of the miR-219 gene in the nervous system and thus suppresses its expression [46]. However, the core enzyme responsible for TCC-induced DNA hypermethylation is not yet defined. Our study found that TCC triggers the hypermethylation of the type II collagen coding gene in a DNMT1-dependent manner in zebrafish and human chondrocytes. Interestingly, our results also suggested that the toxicity of TCC might be heritable. Whether DNMT1-mediated DNA methylation contributes to the heritable toxicity of TCC is worth further investigation.

In conclusion, our study showed that TCC triggers OA in zebrafish anal fins through the induction of DNMT1-mediated hypermethylation of the type II collagen coding gene, which further results in reduced cartilage tissue and narrowed intraarticular space. This articular toxicity of TCC is conserved between zebrafish and human beings. This work is the first discovery of a new toxicity and toxicology of TCC that triggers the occurrence of OA through epigenetic regulation. Furthermore, our findings also highlight a potential therapeutic target for OA.

Environmental implication

Triclocarban (TCC) is one of the antimicrobial agents wildly used for personal care products. It is usually discarded in the wastewater without further processing and thus enriches the environment and our bodies. TCC is currently identified as but not limited to an endocrine disruptor. Our knowledge about the negative impact of TCC on human health is still incomplete. This study first uncovers that TCC triggers osteoarthritis through DNMT1-dependent epigenetic modification. Results of our findings will broaden our understanding of TCC as a hazardous material and thus awaken the potential strategies for substitution or cleanness of TCC.

CRediT authorship contribution statement

Yibo Zhang: Conceptualization, Funding acquisition. Liu He: Data curation, Investigation, Visualization. Yiqi Yang: Investigation, Visualization. Jieqiong Cao: Investigation, Visualization. Zijian Su: Data curation, Investigation. Bihui Zhang: Data curation, Investigation. Huiying Guo: Funding acquisition. Zhenyu Wang: Formal analysis. Peiguang Zhang: Formal analysis. Junye Xie: Formal analysis. Jieruo Li: Resources. Jinshao Ye: Resources. Zhengang Zha: Resources. Hengyi Yu: analyzation of TCC concentration in clinical samples. An Hong: Supervision, Project administration, Funding acquisition, Validation. Xiaojia Chen: Supervision, Project administration, Funding acquisition, Validation.

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.

Acknowledgement

This study was supported by grants from the National Natural Science Foundation of China (No. 81902801), National Natural Science Foundation of China (No. 8217329), National Natural Science Foundation of China (No. 42107451), Guangdong Basic and Applied Basic Research Foundation (No. 2020A1515110534), Fundamental Research Funds for the Central Universities (No. 21620109), Operating Fund of Guangdong Provincial Key Laboratory of Bioengineering Medicine (No. 2014B030301050), and China Postdoctoral Foundation (No. 2019M663375).

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Data availability

Data will be made available on request.

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¹: These authors contributed equally.
这些作者的贡献相同。

[1] [1]
D. Iacopetta, A. Catalano, J. Ceramella, C. Saturnino, L. Salvagno, I. Ielo, et al.
The different facets of triclocarban: a review
Molecules, 26 (2021)
Google Scholar

[2] [2]
Y. Cao, J. Xi, X. You, W. Liu, Y. Luan
Dose-response genotoxicity of triclosan in mice: an estimate of acceptable daily intake based on organ toxicity
Toxicol Res, 10 (2021), pp. 1153-1161
CrossRef View in Scopus Google Scholar

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K. Vimalkumar, E. Arun, S. Krishna-Kumar, R.K. Poopal, N.P. Nikhil, A. Subramanian, et al.
Occurrence of triclocarban and benzotriazole ultraviolet stabilizers in water, sediment, and fish from Indian rivers
Sci Total Environ, 625 (2018), pp. 1351-1360
View PDF View article View in Scopus Google Scholar

[4] [4]
R. Karthikraj, K. Kannan
Mass loading and removal of benzotriazoles, benzothiazoles, benzophenones, and bisphenols in Indian sewage treatment plants
Chemosphere, 181 (2017), pp. 216-223
View PDF View article View in Scopus Google Scholar

[5] [5]
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Research Paper 研究论文Triclocarban triggers osteoarthritis via DNMT1-mediated epigenetic modification and suppression of COL2A in cartilage tissues三氯卡班通过DNMT1介导的表观遗传修饰和软骨组织中COL2A的抑制引发骨关节炎

Highlights 突出

Abstract 抽象

Graphical Abstract 图形摘要

Keywords 关键字

1. Introduction 1. 引言

2. Methods 2. 方法

2.1. Zebrafish line and zebrafish maintenance2.1. 斑马鱼线和斑马鱼维护

2.2. TCC quantification in synovial fluid of OA patients2.2. OA患者滑液中的TCC定量

2.3. Zebrafish imaging 2.3. 斑马鱼成像

2.4. Heartbeat rate 2.4. 心跳率

2.5. Alcian blue and alizarin red double staining2.5. 阿尔新蓝和茜素红双重染色

2.6. Metabolome analysis 2.6. 代谢组分析

2.7. Transcriptome analysis2.7. 转录组分析

2.8. GO analysis 2.8. GO分析

2.9. KEGG analysis 2.9. KEGG分析

2.10. GSEA 2.10. GSEA公司

2.11. Sirius red staining2.11. 天狼星红染色

2.12. Real-time quantified PCR2.12. 实时定量PCR

2.13. Methylation-specific PCR2.13. 甲基化特异性PCR

2.14. Western blotting 2.14. 蛋白质印迹

2.15. Statistical analysis2.15. 统计分析

3. Results 3. 结果

3.1. TCC triggers osteoarthritis in the zebrafish anal fin3.1. TCC引发斑马鱼臀鳍骨关节炎

3.2. TCC programs an osteoarthritis-prone metabolome3.2. TCC对骨关节炎易感代谢组进行编程

3.3. TCC suppresses chondrogenesis and extracellular matrix gene expression

3.4. TCC stimulates DNMT1 expression and methylation of the type II collagen coding gene

3.5. TCC suppresses COL2A1 via DNMT1-dependent DNA methylation

4. Discussion

Environmental implication

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgement

Appendix A. Supplementary material

Data availability

References

Research Paper 研究论文
Triclocarban triggers osteoarthritis via DNMT1-mediated epigenetic modification and suppression of COL2A in cartilage tissues
三氯卡班通过DNMT1介导的表观遗传修饰和软骨组织中COL2A的抑制引发骨关节炎

2.1. Zebrafish line and zebrafish maintenance
2.1. 斑马鱼线和斑马鱼维护

2.2. TCC quantification in synovial fluid of OA patients
2.2. OA患者滑液中的TCC定量

2.5. Alcian blue and alizarin red double staining
2.5. 阿尔新蓝和茜素红双重染色

2.7. Transcriptome analysis
2.7. 转录组分析

2.11. Sirius red staining
2.11. 天狼星红染色

2.12. Real-time quantified PCR
2.12. 实时定量PCR

2.13. Methylation-specific PCR
2.13. 甲基化特异性PCR

2.15. Statistical analysis
2.15. 统计分析

3.1. TCC triggers osteoarthritis in the zebrafish anal fin
3.1. TCC引发斑马鱼臀鳍骨关节炎

3.2. TCC programs an osteoarthritis-prone metabolome
3.2. TCC对骨关节炎易感代谢组进行编程