Integration of Metabolomics and Lipidomics Reveals Metabolic

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Ecotoxicology and Human Environmental Health

Integration of metabolomics and lipidomics reveals metabolic mechanisms of triclosan-induced toxicity in human hepatocytes Hongna Zhang, Xiaojian Shao, Hongzhi Zhao, Xiaona Li, Juntong Wei, Chunxue Yang, and Zongwei Cai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07281 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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Environmental Science & Technology

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Integration of metabolomics and lipidomics reveals

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metabolic mechanisms of triclosan-induced toxicity in

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human hepatocytes

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Hongna Zhang, Xiaojian Shao, Hongzhi Zhao, Xiaona Li, Juntong Wei,

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Chunxue Yang, Zongwei Cai*

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State Key Laboratory of Environmental and Biological Analysis, Department of

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Chemistry, Hong Kong Baptist University, Hong Kong Special Administrative Region

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* Corresponding author (Z.W. Cai)

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Phone: +852-34117070;

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Fax: +852-34117348;

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E-mail: [email protected]

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ACS Paragon Plus Environment


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TOC/Abstract art Lipidomics

Metabolomics

Amino acid metabolism Dysregulated lipids Purine metabolism Energy metabolism Toxicity

OH

Cl O

Oxidative (Decreased in cancer) stress

Cl

Cl

Triclosan

(Increased in cancer) Detoxification O

Cl

HSO3/C6H9O6

O

Cl

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Human liver cells Cl

Triclosan Sulfate/Glucuronide


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ABSTRACT: Triclosan (TCS), an extensively used antimicrobial agent, has raised

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considerable concern due to its hepatocarcinogenic potential. However, previous

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hepatotoxicity studies primarily focused on the activation of specific intracellular

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receptors, the underlying mechanisms still warrant further investigation at the

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metabolic level. Herein, we applied metabolomics in combination with lipidomics to

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unveil TCS-related metabolic responses in human normal and cancerous hepatocytes.

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Endogenous and exogenous metabolites were analyzed for the identification of

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metabolic biomarkers and biotransformation products. In L02 normal cells, TCS

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exposure induced the up-regulation of purine metabolism and amino acid metabolism,

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caused lipid accumulation and disturbed energy metabolism. These metabolic disorders

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in turn enhanced the overproduction of reactive oxygen species (ROS), leading to the

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alteration of antioxidant enzyme activities, down-regulation of endogenous

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antioxidants and peroxidation of lipids. TCS-induced oxidative stress is thus considered

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to be one crucial factor for hepatotoxicity. However, in HepG2 cancer cells, TCS

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underwent fast detoxification through phase II metabolism, accompanied by the

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enhancement of energy metabolism and elevation of antioxidant defense system, which

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contributed to the potential effects of TCS on human hepatocellular carcinoma

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development. These different responses of metabolism between normal and cancerous

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hepatocytes provide novel and robust perspectives for revealing the mechanisms of

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TCS-triggered hepatotoxicity.

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Keywords: triclosan, hepatotoxicity, metabolomics, lipidomics, oxidative stress



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■ INTRODUCTION

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Triclosan [2,4,4´-trichloro-2´-hydroxydiphenyl ether, TCS] has been manufactured

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as an antimicrobial agent for over 50 years. The chemical is extensively employed in

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consumer and personal care products such as toothpastes, soaps, detergents, textiles,

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plastics, and paints.1 The widespread application of TCS has caused contamination and

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potential toxicity in environmental media and biological species.2,3 Beginning in 2017,

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TCS is banned from use in human hygiene biocidal products by the European

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Commission,3 and in over-the-counter consumer antiseptic washing products by the

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U.S. Food and Drug Administration.4 However, these restrictions don’t regulate all

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TCS applications in product formulation. TCS remains one of the most commonly used

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antimicrobials worldwide, its human health risks still warrant further evaluation.

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Humans are exposed to TCS mainly through dermal contact with personal care

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products and devices,5 as well as ingestion of contaminated food and drinking water.6,7

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The frequent exposure of TCS has resulted in its wide detection in human body fluids,

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including blood, urine, amniotic fluid, and human milk.8-11 The epidemiologic study

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has demonstrated associations between TCS exposure and allergic sensitization.12

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Through in vitro and/or in vivo models, TCS was found to be implicated with endocrine

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disruption,13 impaired muscle contraction14, and carcinogenesis.15,16 In vivo studies

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showed that TCS could cause liver cell proliferation and fibrotic responses, functioning

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as a liver tumor promoter.15,17 The effects of liver tumor promotion has been attributed

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to the activation of the nuclear receptor constitutive androstane receptor (CAR) through

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xenobiotic receptor screening assay.15 Moreover, Ashrap et al.18 found that TCS could


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be transformed into more lipophilic and bioactive metabolites with much higher CAR

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activation than the parent compound in humans. As one pathogenic mechanism

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associated with liver fibrosis, oxidative stress is suspected as one of the underlying

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factors for TCS-mediated liver carcinogenesis.19,20 Ma et al.21 demonstrated that TCS

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treatment in human cancer cells significantly reduced the levels of global DNA

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methylation, which is considered as a biomarker of cancer progression. However, the

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links between TCS-medicated receptor activation and physiological effects of

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hepatotoxicity remain largely unknown, and need to be further identified through the

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evaluation of downstream metabolic processes.

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Mass spectrometry (MS)-based metabolomics is an efficient strategy in toxic risk

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assessment by monitoring small molecule metabolites within biological systems in

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response to external stressors.22 As a branch of metabolomics, lipidomics is also a

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powerful method for integral investigation of biological responses based on the lipid

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profiling in biological samples.23 Up to now, TCS-triggered responses of hepatocellular

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endogenous metabolites have not been well elucidated. The combined application of

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metabolomics and lipidomics is expected to promote comprehensive and deep

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understanding of the metabolic mechanisms of TCS-induced hepatotoxicity.

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Biotransformation is an organism’s important defense against xenobiotics. TCS is

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generally considered to be rapidly detoxified through glucuronidation and sulfonation

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by phase II metabolism in humans.24 To accurately comprehend the liver toxicity of

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TCS, it is also essential to evaluate TCS biotransformation in biological models.

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Therefore, the objective of this study was to explore the metabolic mechanisms of



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TCS-induced hepatotoxicity, especially the carcinogenic potential. The human normal

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hepatocyte L02 cell line and the hepatocarcinoma HepG2 cell line were chosen as the

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model systems. These two cell models have been widely applied for hepatotoxicity

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studies of xenobiotics to predict the potential human health risks.25,26 L02 hepatocytes

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were selected due to the normal liver-specific functions. Enhanced cell proliferation

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was observed in HepG2 cells after TCS treatment,15 it is thus desirable for the

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mechanism investigation of TCS effects on the human hepatocellular carcinoma (HCC)

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development. We firstly performed TCS impact on cell viability to explore its

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cytotoxicity. MS-based metabolomics and lipidomics approaches were applied to

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evaluate the cellular metabolic responses to TCS exposure. To achieve a better

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understanding of the involved toxic mechanisms, the biotransformation of TCS was

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also investigated. The reactive oxygen species (ROS) generation and anti-oxidative

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responses in TCS-treated cells were further determined to evaluate the oxidative

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stresses. The obtained results were expected to provide new insights into the

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mechanisms of TCS-induced hepatotoxicity by comparing the metabolic differences in

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normal and cancer liver cells.

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■ MATERIALS AND METHODS Chemicals and Materials. TCS was purchased from TCI (purity ≥ 98.0%, Tokyo 13C

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Chemical Industry, Japan). Stable isotope-labeled standard of

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Wellington Laboratories Inc. (Guelph, Ontario, Canada). Dulbecco's modified eagle

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medium (DMEM) high glucose cell culture medium, fetal bovine serum (FBS),


12-TCS

was from

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penicillin-streptomycin, 0.25% trypsin-EDTA, and phosphate-buffered saline (PBS)

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were from GibcoTM (Thermo Fisher Scientific, Waltham, MA). All the other chemicals

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used were of analytical grade or better. Solvents, including methanol (MeOH),

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acetonitrile (ACN), isopropanol (IPA), and methyl tert-butyl ether (MTBE) were of

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HPLC grade (Duksan, Seoul, Korea). Purified water obtained by a Millipore water

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purification system (Synergy® UV, Bedford, MA) were used throughout the experiment.

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Cell Culture and Cell Viability Assay. Human normal hepatic cell line L02, and

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human hepatocarcinoma cell line HepG2 were purchased from the Shanghai Cell Bank

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of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured

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in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin, and

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maintained at 37℃ in a humidified atmosphere of 5% CO2. To examine cell viability,

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L02 and HepG2 cells were seeded at a density of 2 × 104/well in 96-well plates. After

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attachment for 24 h, the cells were exposed to TCS in serum-free DMEM containing

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1% penicillin-streptomycin for 48 h. The exposure groups were implemented with 0,

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0.01, 0.1, 0.5, 1, 2.5, 5, 10, 15, and 20 μM TCS dissolved in DMSO (0.05%, v/v). Cell

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viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation

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Assay (Promega, Madison, WI). The solution reagent contains a tetrazolium compound

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of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphenyl)-

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2H-tetrazolium, inner salt]. At post-treatment time, 20 μL of the MTS solution reagent

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was added to 100 μL of culture media in each well and incubated for 2 h at 37℃. The

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absorbance was recorded at 490 nm using a Victor X3 multilabel plate reader (Perkin

131

Elmer, Waltham, MA). The results were expressed as a percentage of cell viability



132

normalized to the control group.

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Metabolomics Sample Preparation and Metabolite Extraction. Ten replicates

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were performed for the control and TCS-treated groups. For each sample, 1 × 106 cells

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were seeded in the 6-cm dish in fresh medium for 24 h. The cells were then exposed to

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TCS in serum-free medium based on the cell viability results (0, 1, 2.5 μM for L02

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cells; 0, 1, 10 μM for HepG2 cells). After 48 h, the culture medium was removed, the

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cells were quickly rinsed with PBS, and harvested by trypsin digestion. Cell pellets

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collected from each dish were rapidly quenched by 500 μL of chilled MeOH/H2O (4:1,

140

v/v) and conserved in liquid nitrogen in 2.0 mL Eppendorf tubes. The cell disruption

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was conducted by freeze-thawing with liquid nitrogen.

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For the metabolite extraction, the supernatants were collected after sample

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centrifugation (15,000 g, 10 min, 4℃), and dried at 4°C using a Max-Up (NB-504CIR)

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IR vacuum concentrator (N-Biotek Inc., GyeongGi-Do, Korea). The residuals were re-

145

dissolved in MeOH/H2O (1:1, v/v) which contained 1 μg/mL of 4-chloro-phenylalanine

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as the internal standard (IS), the solvent amount was proportional to cell number that

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was counted in prior. The samples were vortexed, and followed by centrifugation

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(15,000 g, 10 min, 4℃) for supernatant collection. The quality control (QC) sample was

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prepared by pooling 20 μL of solution from each sample in all groups. For the extraction

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of extracellular TCS and its biotransformation products, the culture medium from each

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dish was transferred into a 2.0 mL Eppendorf tube and centrifuged at 1,000 g for 5 min.

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Supernatant (500 μL, containing 10 ng of 13C12-TCS) was then extracted as described

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in the Supporting Information (SI) before the analysis of TCS biotransformation


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products.

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Lipidomics Sample Preparation and Lipid Extraction. The sample preparation

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method of lipidomics was the same as that of metabolomics as described above. After

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the cell disruption, lipids were extracted based on the method of Matyash et al.27 with

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some modifications. Briefly, 1.2 mL of MTBE was added to the disrupted cells,

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followed by vortexing extraction for 10 min. Phase separation was then induced by

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adding 200 μL of water. After 10 min of incubation at room temperature, the samples

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were centrifuged (15,000 g, 10 min, 4 ℃) for organic phase collection. The lower phase

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was re-extracted with 400 μL of the solvent mixture, which obtained by mixing

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MTBE/MeOH/water (12:4:3, v/v/v) and collecting the upper phase. The combined

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organic phases were dried at 4℃ using the vacuum concentrator and stored at −80℃.

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Before instrumental analysis, the residuals were reconstituted in ACN/IPA/H2O

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(65:30:5,

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phosphocholine as the IS, the solvent amount was proportional to cell number that was

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counted in prior. The supernatant was then collected after sample vortex and

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centrifugation (15,000 g, 10 min, 4℃). The QC sample was prepared by mixing 20 μL

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of solution from each sample in all groups.

v/v/v)

containing

5

μg/mL

of

1,2-dinonadecanoyl-sn-glycero-3-

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Instrumental Analysis. The metabolimics samples were analyzed using a Thermo

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Scientific ultra-high performance liquid chromatography system (UHPLC) coupled to

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a Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (QE Orbitrap

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MS). The instrumental conditions were modified according to the method of Xiang et

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al.28 The lipidomics samples were analyzed using a Thermo Scientific UHPLC coupled



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to an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Orbitrap Fusion MS). The

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instrumental conditions were optimized based on the method of Bird et al.29 Details of

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chromatographic conditions and MS parameters are summarized in Tables S1 and S2.

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Samples of the control and TCS-treated groups were analyzed successively. The blank

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and QC samples were injected at the beginning and at the end of the sample run, the

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QC sample was also analyzed every five samples.

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Data Processing and Analysis. The Xcalibur Software v.4.1 (Thermo) was used for

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data acquisition and pretreatment. For the metabolimics samples, chromatography peak

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alignment and metabolite extraction were achieved using the software R. For lipid peak

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acquisition and alignment, the software LipidSearch (Thermo) was utilized. Detailed

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parameters of R package and LipidSearch are available in the SI. Metabolic features

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with disturbed signals in blank samples and coefficient of variation (CV) > 30% in QC

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samples were excluded to eliminate the potential contamination. The qualified

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metabolic peaks were imported into software SIMCA-P (Version 13.0, Umetrics, Umea,

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Sweden) for the partial least squares discriminant analysis (PLS-DA). The

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differentiating metabolites between the control and exposure groups were selected

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based on the variable importance in projection (VIP) scores in PLS-DA (VIP > 1), as

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well as P-value (P < 0.05) and fold change (FC, FC >1.2 or < 0.8). The P values were

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calculated in comparison of the control using Student’s t-test. Metabolites were

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structurally identified by matching MS/MS fragments with data in the databases of

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METLIN (http://metlin.scripps.edu/) and Human Metabolome Database (HMDB,

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http://www.hmdb.ca/), and the software Lipidsearch. Metabolite biomarkers were


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further confirmed through the comparison of authentic standards. Pathway analysis

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based on the identified metabolites was carried out using MetaboAnalyst according to

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the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database

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(www.genome.jp/kegg/).

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TCS Biotransformation Product Identification and Quantification. According to

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metabolomics data operated in negative ionization mode, the biotransformation

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products of TCS in cell extracts were identified based on the absence of peak intensity

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in control group but with abundance in exposure groups. Another important feature is

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the exhibition of several peak clusters in MS due to the presence of chlorine atoms.

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Their structures were further elucidated through MS/MS analysis. Standards of

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triclosan sulfate (TCSS) and triclosan glucuronide (TCSG) were synthesized as

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described in the SI, the 1H-NMR of TCSS is shown in Figure S1. TCS, TCSS and TCSG

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in the samples were quantified using a Thermo Scientific UHPLC coupled to a TSQ

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Quantiva™ Triple Quadrupole Mass Spectrometer (UHPLC–MS/MS). Electrospray

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ionization was operated in negative mode, and multiple reaction monitoring (MRM)

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was used for the analysis. The hydroxylated metabolites of TCS were identified by

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UHPLC–Orbitrap Fusion MS. Detailed information of the above instrumental methods

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are shown in Tables S3 and S4. Quality control and quality assurance are provided in

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the SI.

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Relevant Metabolic Enzyme Activities and Intracellular Ammonia Level. To

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further explore the toxicity mechanisms, TCS-treated cells were lysed, then the

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supernatant was collected after centrifugation (15,000 g, 5 min, 4℃) for further analysis.



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The specific assay kits were used to measure the activities of xanthine oxidase (XOD,

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Nanjing Jiancheng Bioengineering Institute, China) and succinate dehydrogenase

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(SDH, Sigma-Aldrich, Milwaukee, WI), following the manufacturer instructions. The

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results were normalized to the protein content. For the evaluation of ammonia

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production, TCS-treated cells were rinsed with PBS and snap frozen in liquid nitrogen.

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Intracellular ammonia was quantified with the Ammonia Assay Kit (Sigma-Aldrich

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Chemical, Milwaukee, WI) following the recommended manufacturer’s protocol.

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ROS Detection and Oxidative Stress Marker Determination. Cellular production

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of ROS was measured using the molecular probe 2′,7′-dichlorodihydrofluorescein

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diacetate (H2DCF-DA, Life technologies, Eugene, OR). After TCS exposure for 48 h,

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L02 and HepG2 cells were incubated with 10 µM H2DCF-DA for 30 min at 37°C, then

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washed three times with serum-free medium before the fluorescence record.

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Fluorescence intensities were analyzed by the software ImageJ (National Institutes of

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Health, Bethesda, MD). For the determination of other oxidative stress markers, the

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specific assay kits (Beyotime Institute of Biotechnology, Beijing, China) were adopted

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to determine the level of malondialdehyde (MDA), and the activities of superoxide

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dismutase (SOD) and catalase (CAT) in cell lysate. The assays were performed

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according to the manufacturer instructions, and the determinations were normalized to

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the protein content. The relative contents of glutathione (GSH) and taurine were

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calculated according to the metabolomics results.

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■ RESULTS AND DISCUSSION


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Cytotoxicity Assay. After 48 h of incubation with TCS, cell viabilities were

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determined to obtain the optimal exposure concentrations for metabolomics and

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lipidomics studies. No difference in cell viability was observed with FBS

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concentrations ranged 0–10% for L02 and HepG2 hepatocytes (Figure S2), thus serum

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free medium was used to eliminate the combination of TCS with proteins in FBS. As

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shown in Figure 1A, the toxicity of TCS was unobvious for both hepatocytes when TCS

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concentration was less than 1 μM. Then L02 cell viability decreased significantly in a

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dose-dependent manner. A decrease in HepG2 cell viability was observed at

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concentrations above 5 μM. We chose 2.5 μM for L02 cells and 10 μM for HepG2 cells

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as high-dose exposure concentrations, because the corresponding cell viabilities were

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around 80% for both cell types. Besides, 1 μM of TCS was set as the low-dose exposure

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concentration. It should also be noted that the median lethal dose (LD50) of TCS in

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normal L02 cells was about 5 μM (Figure 1A). This TCS value and all our treatment

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levels were within those detected in human urine, which have been reported to contain

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TCS as high as 13 μM.30,31

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Potential Metabolite Biomarkers and Perturbed Metabolic Pathways. Global

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metabolomics was applied to profile the metabolic changes of hepatic cells in response

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to TCS exposure. The PLS-DA models demonstrated satisfactory explanation, fitness,

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and prediction power for L02 cells (Figure 1B-C) and HepG2 cells (Figure 1E-F).

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Distinct differences were observed between the control group and TCS-treated groups,

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suggesting that TCS induced conspicuous perturbation of intracellular metabolites. The

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volcano plot screening showed that TCS affected the hepatic cells in a dose-dependent



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manner (Figure S3). For the same TCS concentration of 1 μM, more metabolite features

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were significantly changed in L02 cells than in HepG2 cells (Figures S3A and S3C),

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indicating that TCS induced more severe alterations of metabolic profiles in normal

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hepatocytes. This result was consistent with the higher cytotoxicity of TCS in L02 cells.

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Among the significantly changed endogenous metabolites, 42 of them in L02 cells and

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45 in HepG2 cells were structurally identified, with 22 metabolites in common (Tables

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S5 and S6). Those biomarkers mainly included amino acids, peptides, nucleosides,

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nucleotides, carboxylic acids, phospholipids, and acylcarnitines. Based on the KEGG

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pathway database, the significantly changed metabolites upon TCS intervention were

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highly associated with the perturbation of alanine, aspartate and glutamate metabolism;

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purine metabolism; and tricarboxylic acid (TCA) circle in both hepatocytes. In addition,

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taurine and hypotaurine metabolism in L02 cells, and glutathione metabolism in HepG2

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cells were also significantly impacted by TCS exposure (Figures 1D and 1G). The

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details regarding pathway perturbation were further discussed as follows.

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TCS Exposure Associates with the Disruption of Amino Acid Metabolism and

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Purine Metabolism. In normal and cancerous hepatocytes, the most relevant metabolic

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pathway affected by TCS exposure was alanine, aspartate and glutamate metabolism.

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The contents of downstream metabolites in this pathway were significantly up-

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regulated (Figure 2). In comparison with the high-dose and control groups, the FCs of

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pyruvate, fumarate, and succinate in L02 cells were 1.50, 2.00, and 1.76, respectively.

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Elevated levels of pyruvate (FC 1.44), fumarate (FC 1.92), and α-ketoglutarate (FC

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2.46) were observed in HepG2 cells. These results revealed the possible up-regulation


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of this metabolic pathway. It should be mentioned that the above metabolites could also

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be generated during other amino acid metabolism. Moreover, these metabolic

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intermediates can also serve as a source of carbon skeletons for the synthesis of amino

289

acids. The significantly changed metabolite biomarkers were also involved in

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phenylalanine metabolism, cysteine and methionine metabolism, and tyrosine

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metabolism for both hepatocytes (Tables S5 and S6). Therefore, all 20 amino acids

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were further selected and structurally identified using the standards (Figures S4 and

293

S5). According to their FCs, the intracellular contents of 17 amino acids decreased in

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TCS-treated L02 cells with 10 of them significantly decreased in dose-dependent

295

manner, indicating the acceleration of amino acid metabolism. Additionally, 12 amino

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acids showed decreasing tendency for low or/and high TCS exposure in HepG2 cells,

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these may due to the promoted import of extracellular nutrients into the cancer cells.

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Oxidative deamination of amino acids in the liver is the main source of endogenous

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ammonia. The generation of ammonia occurs mainly via the α-amino nitrogen of

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glutamate. Glutamate levels decreased in L02 cells and increased in HepG2 cells after

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TCS treatment (Figures S4 and S5), indicating the possible disturbance of ammonia

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formation. Alanine production through partial amino acid catabolism is an important

303

indicator for ammonia detoxification.26 The down-regulation of alanine in liver cells

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implied the potential accumulation of ammonia. Another important evidence for the

305

overproduction of ammonia is the elevated levels of N-acetylglutamate, which is an

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essential allosteric activator of carbamoyl phosphate synthetase I (CPS1), the rate-

307

limiting enzyme of the urea cycle (Figure S6A). When the hepatic ammonia



308

concentration tends to increase, it would activate glutaminase to enhance

309

intramitochondrial N-acetylglutamate level; these in turn, by activating CPS1, will

310

actually convert ammonia to urea for excretion.32 In this study, TCS treatment induced

311

significant increase of N-acetylglutamate in L02 cells (FC 1.49) and HepG2 cells (FC

312

5.98. Moreover, through the quantification of ammonia levels, we directly

313

demonstrated the elevation of intracellular ammonia in TCS-treated cells (Figure S6B).

314

These results clearly showed that TCS exposure led to the up-regulation of amino acid

315

metabolism in human hepatocytes.

316

TCS intervention was also considered to be highly responsible for the perturbation

317

of purine metabolism in normal and cancerous hepatocytes. Purine metabolism is at the

318

downstream of nucleic acid degradation. In TCS-treated L02 cells, all the identified

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downstream intermediate levels significantly increased (Figures 2A and 2B),

320

suggestive of the up-regulation of this metabolic pathway. It should be stressed that,

321

hypoxanthine and xanthine are the substrates of XOD which produces hydrogen

322

peroxide (H2O2). XOD activity in L02 cells was significantly enhanced after high-dose

323

TCS exposure (Figure 2A). Therefore, the up-regulation of purine metabolism was a

324

critical source of ROS in normal hepatocytes. However, in TCS-treated HepG2 cells,

325

the concentration of hypoxanthine decreased, while no significant change was observed

326

for xanthine (Figures 2A and 2C).

327

TCS Exposure Associates with Lipid Metabolism Perturbation. It’s noteworthy

328

that some metabolites were not listed under the metabolic pathway database. The levels

329

of several acylcarnitines, including palmitoylcarnitine (C16), tetradecanoylcarnitine


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(C14), 2-methylbutyroylcarnitine (C5) in L02 cells, and isobutyrylcarnitine (C4) in

331

HepG2 cells drastically increased after TCS exposure (Tables S5 and S6, Figure S7).

332

The accumulation of even-chain C14 and C16 was associated with incomplete fatty

333

acid β-oxidation within the mitochondrial matrix, odd-chain C5 arose from amino acid

334

catabolism, whereas C4 could be produced from both fatty acids and amino acids.33

335

Additionally, three metabolites in L02 cells, and five in HepG2 cells were involved in

336

the glycerophospholipid metabolism. Glycerophosphorylcholine is one form of choline

337

storage in the cytosol, its content significantly decreased in TCS-treated liver cells.

338

Choline, the main source of phosphatidylcholines (PCs), showed opposite change

339

tendency between L02 and HepG2 cells. As the oxidative metabolites of PCs, LPC(16:0)

340

was up-regulated in L02 cells, while LPC(14:0) and LPC(16:0) were down-regulated

341

in HepG2 cells. Same trends were observed for LPEs, the breakdown products of

342

phosphatidylethanolamines (PEs). Changes of these biomarkers demonstrated that TCS

343

induced intercellular lipid dysregulation, the malfunction between normal and

344

cancerous hepatocytes could be different.

345

These abnormalities of lipid metabolism were further investigated through

346

lipidomics analysis. PLS-DA score plots and volcano plots showed TCS treatment

347

induced significant perturbation of intracellular lipids (Figure S8). Abundance of the

348

identified lipids displayed significant up-regulation in triglyceride (TG), diacylglycerol

349

(DG), PC, PE, phosphatidylglycerol (PG), LPC, LPE, ceramide (Cer), and

350

sphingomyelin (SM) as well as remarkable down-regulation in polyphosphoinositide

351

(PI) in L02 cells (Figure S9A). The subclass lipids of DG, PC, PE, and SM significantly



352

increased, whereas TG, PG, PI, LPC, LPE, and Cer decreased in HepG2 cells (Figure

353

S9B). The lipid biomarkers were further selected based on VIP scores, P-value and

354

FCs. The results are shown in the heat maps of Figure 3. Of note, the change trends of

355

LPC and LPE were consistent with those obtained from metabolomics. It should also

356

be pointed out that PC (16:0/16:0) was up-regulated in both cells, showing different

357

change tendency from its precursor LPC(16:0).

358

In TCS-treated L02 cells, the lipid biomarkers were generally up-regulated except

359

several individual PCs (Figure 3A). A dose-dependent increase of TGs occurred,

360

indicating that TCS induced TG accumulation in normal hepatocytes, which is a

361

hallmark feature of the pathogenesis of nonalcoholic fatty liver disease in humans.34 As

362

two main components of phospholipids, PC and PE contents were significantly

363

enhanced, indicating the disruption of cell membrane homeostasis. It should be noted

364

that the PC class showed an increase trend although several individual PCs were down-

365

regulated (Figure S9A and 3A), this could result from the up-regulation of the more

366

abundant PCs. LPC level was dramatically up-regulated, serving as an important

367

evidence of the hepatic lipotoxicity.35 PG is a precursor for the synthesis of cardiolipin,

368

an important component of the inner mitochondrial membrane. The remarkable

369

increase of PG levels suggested that TCS treatment disturbed the structure of

370

mitochondrial membrane. The SM cycle, specifically the conversion of SM to Cer, has

371

been regarded as a key signaling pathway involved in apoptosis. In this study, TCS-

372

associated up-regulation of Cer was an important signal for the activation of apoptotic

373

cascade. Moreover, high abundance of Cer could enhance the production of ROS by


Page 18 of 38

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374

directly acting on the mitochondrial respiratory electron chain.36 Therefore, TCS

375

treatment triggered lipid deposition in human normal hepatocytes. This result was

376

consistent with those obtained in zebrafish embryos and tadpole hepatocytes, which

377

TCS induced lipid droplet accumulation through the reduction of fatty acid β-

378

oxidation.37,38 In TCS-treated HepG2 cells, the depletion of TG could arise from the

379

increased energy demand, and/or the decreased fatty acid synthesis. Moreover, defects

380

on Cer generation and SM metabolism were also observed (Figure S9B, Figure 3B). It

381

has been reported that malignant cells with low Cer levels are resistant to apoptosis,

382

and Cer content in cancer cells might be involved in the pathogenesis of tumor growth.39

383

In our study, the decrease of Cer level in HepG2 cells provided an evidence for the

384

apoptotic resistant features in response to TCS exposure, which might contribute to

385

TCS-associated human HCC development.

386

TCS Exposure Associates with Energy Metabolism Disruption. Based on the

387

metabolomics analysis, disorders of energy metabolism were observed in TCS-treated

388

hepatocytes (Figure 4). Glucose was supplied in cell culture media as the major energy

389

source, it could be converted into pyruvate and subsequently lactate by the pathway of

390

glycolysis. In L02 and HepG2 cells, pyruvate and lactate levels presented increasing

391

tendencies with the increase of TCS doses, suggestive of the up-regulation of glycolysis

392

in the cytoplasm. It is important to note that, in oncology, the majority of cancer cells

393

have been observed to predominantly produce energy by glycolysis.40,41 Another key

394

feature of the metabolic profile of cancer cells is the increased glutaminolysis for energy

395

production.42,43 Elevated levels of glutamine and glutamate were observed in TCS-



396

treated HepG2 cells (Table S6 and Figure S5), indicating the energy production from

397

glutamine metabolism should also be enhanced. Therefore, TCS-induced energy

398

enhancement may contribute to the promotion of cancer cell proliferation. Additionally,

399

as discussed above, TCS caused a significant acceleration of amino acid metabolism

400

and disturbance of fatty acid β-oxidation in liver cells. Pantothenic acid is a component

401

of the acyl group carrier coenzyme A (CoA-SH), its intracellular contents significantly

402

increased after TCS exposure. These changes could elevate the generation of acetyl-

403

CoA, which was then oxidized in the TCA cycle.

404

Mitochondrial TCA cycle is the hub of cellular energy metabolism. Constituents of

405

the TCA cycle, including citrate, fumarate, and malate were significantly up-regulated

406

in TCS-treated hepatocytes. In addition, the level of succinate increased in L02 cells,

407

and α-ketoglutarate increased in HepG2 cells. Metabolite biomarkers observed in L02

408

cells also include flavin adenine dinucleotide (FAD) and nicotinamide adenine

409

dinucleotide (NAD+). Playing vital roles in energy metabolism, NAD+ and FAD accept

410

hydride equivalents to generate reducing equivalents, NADH and FADH2, respectively,

411

which are then furnished to the mitochondrial electron transport chain to fuel oxidative

412

phosphorylation for energy production. It has been extensively proved that TCS is a

413

mitochondrial uncoupler that disrupted membrane potential and inhibited the activity

414

of SDH (complex II), resulting in the enhancement of ROS production.44-46 In this study,

415

the decreases of SDH activity in TCS-treated cells were also observed (Figure S10). In

416

addition, lipidomics analysis demonstrated that TCS treatment disturbed the

417

mitochondrial membrane homeostasis. Succinate, the substrate of complex II,


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418

significantly increased in L02 cells, which could result from the complex II activity

419

changes and enhanced energy metabolism. Moreover, succinate has been identified as

420

an oncometabolite, its accumulation in mitochondria is considered to be associated with

421

tumor progression.47 Mitochondria ROS generated at the electron transport chain

422

during oxidative phosphorylation is the major source of the cellular oxidative

423

burden.48,49 Therefore, TCS-induced up-regulation of TCA cycle could lead to the

424

accumulation of mitochondrial ROS, which further enhanced oxidative stresses in

425

normal hepatocytes.

426

Biotransformation of TCS in Hepatic Cells. After exposure for 48 h, TCS was

427

detected in L02 cells, with the FC of 3.48 between high and low exposure groups (Table

428

1, Figure S11). However, TCS content was below the detection limit in HepG2 cells,

429

even that the high-dose exposure group was in the initial concentration of 10 μM. In

430

the culture medium, the mass composition of TCS was 12.7% in low and 10.2% in high

431

exposure groups for L02 cells, while only about 1% of TCS remained for HepG2 cells

432

(Figure S12). These results demonstrated the fast biotransformation of TCS in

433

cancerous hepatocytes. In cell extracts, sulfate and glucuronide conjugated metabolites

434

of TCS were identified (Table 1). The structures of TCSS and TCSG were further

435

elucidated and confirmed using the synthesized standards (Figures S13 and S14). In

436

L02 cells, FCs (High/Low) of TCSS and TCSG were 3.79 and 7.54, respectively, this

437

could be explained by the preference generation of TCSG than TCSS in humans.24 After

438

TCS exposure, intracellular and extracellular TCSS and TCSG were further monitored

439

using UHPLC-MS/MS. TCSG/TCSS peak area ratio in the extracellular culture media



440

was much higher than that in intracellular extracts for both hepatocytes (Figure S15),

441

demonstrating that TCS was mainly transformed into TCSG and excreted by the liver

442

cells. Moreover, TCSG/TCSS peak area ratio in culture media of HepG2 cells was

443

much higher than that in L02 cells (> 15 folds), further demonstrating the rapid phase

444

II metabolism of TCS in cancerous hepatocytes. Therefore, the lower toxicity of TCS

445

to HepG2 cells was partly attributed to its fast biotransformation into conjugated

446

metabolites, and excretion for detoxification.

447

It should be pointed out that the upstream target of TCS has been demonstrated to be

448

the nuclear receptor CAR, the regulated genes of which are members of the CYP2B,

449

CYP2C, and CYP3A subfamilies.15,18 Under the catalysis of these cytochrome P450

450

enzymes, TCS underwent the rapid phase I metabolism to form hydroxylated

451

metabolites (OH-TCS).18 In this study, no OH-TCS was detected in the samples after

452

48 h of TCS exposure. However, sulfate and glucuronide conjugates of OH-TCS, OH-

453

TCSS and OH-TCSG, were identified in both extracellular and intracellular extracts

454

(Figure S16), indicating the generation of OH-TCS. Furthermore, the structure of OH-

455

TCS was elucidated in the extracts of hepatic cells treated with TCS for 3 h, directly

456

demonstrating the fast phase I metabolism of TCS (Figure S17). Formation of ROS, in

457

particular, superoxide anion radical and H2O2, can emerge following the breakdown or

458

uncoupling of the P450 catalytic cycle.49,50

459

ROS Generation and Oxidative Stress Markers. Based on above analysis, we

460

hypothesized that ROS was excessively generated within the hepatic cells, altered the

461

intracellular redox status and exacerbated metabolic dysfunction. To verify the


Page 22 of 38

Page 23 of 38


462

overproduction of ROS, real-time generation of ROS was imaged using the fluorescent

463

probe of H2DCF-DA. The fluorescence signal intensities of TCS-treated groups were

464

significantly stronger than that of the control group in L02 and HepG2 cells (Figure

465

5A), demonstrating that TCS induced the excessive accumulation of ROS and created

466

a prooxidant environment in hepatocytes. This result was consistent with those obtained

467

in mouse models.15 MDA is the end product of lipid peroxidation and can serve as a

468

sensitive diagnostic index of oxidative injury. MDA content in TCS-treated L02 cells

469

was significantly elevated in a concentration-dependent manner, similar to the trends

470

of ROS accumulation within the cells (Figure 5B). These results provide solid evidence

471

that TCS treatment led to oxidative stresses and lipid peroxidation in normal

472

hepatocytes. However, TCS exposure did not induce MDA content changes in HepG2

473

cells (P > 0.05, Figure 5B), which indicated that serious oxidative damage did not occur

474

in cancerous hepatocytes. As for the antioxidant enzymes, SOD activities increased,

475

while CAT activities decreased in response to TCS exposure in both liver cells (Figures

476

5C and 5D), which could lead to the intracellular accumulation of H2O2. GSH and

477

taurine are endogenous antioxidants that protect against oxidative stress in liver,50,51

478

their contents significantly decreased in TCS-treated L02 cells (Figures 5E and 5F).

479

These results clearly demonstrated a dose-dependent variation of cellular oxidative

480

status in normal liver cells. In HepG2 cells, GSH content was dramatically elevated,

481

indicating the enhancement of anti-oxidative defense system in response to TCS

482

treatment. According to the metabolic pathway analysis, TCS induced significant up-

483

regulation of glutathione metabolism in HepG2 cells (Figure 1G), suggestive of a



484

positive anti-oxidative response. Therefore, TCS-induced oxidative stress was one

485

crucial factor that caused cytotoxicity in normal hepatocytes. The elevation of anti-

486

oxidative defense system in TCS-treated cancer cells contributed to the HCC

487

development.

488

Insights into Toxicity Mechanism. This study provides direct evidence for the

489

metabolic mechanisms of toxicity induced by environmental dose-related TCS in

490

human hepatocytes. On the basis of the obtained results, the possible mechanisms of

491

TCS hepatotoxicity are deduced as follows. As a xenobiotic, TCS activates the nuclear

492

receptor CAR and is metabolized by cytochrome P450 enzymes,15,18 which triggers the

493

oxidative stress. As the adaptive responses, purine metabolism and amino acid

494

metabolism are up-regulated, lipid metabolism and energy metabolism are disturbed.

495

These metabolic disorders in turn enhance the overproduction of ROS mainly from

496

XOD activity and mitochondrial dysfunction, then exacerbate oxidative damage. The

497

potential mechanisms of TCS effects on the human HCC development include the up-

498

regulation of energy metabolism, elevation of antioxidant defense system, and fast

499

detoxification through phase II metabolism. Moreover, this study also showed that

500

metabolomics is a powerful strategy for the simultaneous identification of endogenous

501

and exogenous metabolites in biological samples. The information presented here is

502

useful to improve current understanding of TCS-induced hepatotoxicity. Future

503

research will include TCS-related metabolomics study in human body fluids for further

504

mechanism elucidation.

505


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506

■ ASSOCIATED CONTENT

507

Supporting Information

508

Additional information as noted in the text. This material is available free of charge via

509

the Internet at http://pubs.acs.org.

510

■ AUTHOR INFORMATION

511

Corresponding Author

512

* Phone: +852-34117070. Fax: +852-34117348. E-mail: [email protected].

513

Notes

514

The authors declare no competing financial interest.

515

■ ACKNOWLEDGMENTS

516

We thank the National Natural Science Foundation of China (21777010 and 21806134),

517

and General Research Fund (12301518) and Collaborative Research Fund (C2014-14E)

518

of Hong Kong Research Grants Council for financial support.

519

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(50) Valko, M.; Rhodes, C. J.; Moncol, J.; Izakovic, M.; Mazur, M. Free radicals,

668

metals and antioxidants in oxidative stress-induced cancer. Chem-Biol. Interact. 2006,

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(51) Bouckenooghe, T.; Remacle, C.; Reusens, B. Is taurine a functional nutrient? Curr. Opin. Clin. Nutr. Metab. Care 2006, 9 (6), 728–733.


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Page 33 of 38

672


Table 1. TCS and its phase II metabolites identified in cell extracts based on the metabolomics data Compound RT (min) TCS

16.13

TCSS

13.80

TCSG

11.99

Major measured mass (m/z)

Molecular structure

∆ppm

Mass pattern

FC (L02) High/Low a

FC (HepG2) High/Low a

286.9440 288.9412 366.9007 368.8976 462.9761 464.9728

[C12H6O235Cl3][C12H6O235Cl237Cl][C12H6SO535Cl3][C12H6SO535Cl237Cl][C18H14O835Cl3][C12H14O835Cl237Cl]-

0.35

–b –b 286.9438 288.9410 286.9440, 175.0239, 113.0228 288.9406, 175.0239, 113.0229

3.48 3.48 3.79 3.79 7.54 7.00

–c –c 11.4 11.0 7.14 7.17

0.01 0.22

673

a Peak

674

b

No high abundant product ion was observed within the detection range (> m/z 50) of our instrument, TCS was confirmed by the RT with standard.

675

c

No signal was observed.

intensity ratio between high/low exposure groups, the theoretical value was 2.5 for L02 cells and 10 for HepG2 cells.



676

Page 34 of 38

FIGURES (B)

Control

1μM

2.5μM

QC

(C)

Control

2.5μM

1μM

QC

(D)

Alanine, aspartate and glutamate metabolism

Negative mode (–)

Positive mode (+)

(A)

OH

Cl

TCA cycle

O Cl

Cl

Taurine and hypotaurine metabolism

L02

Purine metabolism

TCS 120

L02 HepG2 



80 60 40

 

(E)

Control

1μM

10μM

QC

Control

1μM

10μM

QC

Negative mode (–)

Positive mode (+)



(F)

(G) Alanine, aspartate and glutamate metabolism

20 0

TCA cycle

 

Purine metabolism

0 0.01 0.1 1 2.5 5 10 15 20

Glutathione metabolism

TCS concentration (M)

R2X = 0.559, R2Y = 0.879, Q2 = 0.832

HepG2

Cell viability (%)

R2X = 0.555, R2Y = 0.900, Q2 = 0.873

R2X = 0.461, R2Y = 0.817, Q2 = 0.769



100

R2X = 0.725, R2Y = 0.869, Q2 = 0.832

677 678

Figure 1. (A) The cell viability of L02 and HepG2 hepatocytes after TCS exposure. Data are the mean ± SD of n = 6, * P < 0.05, ** P < 0.01 (in

679

comparison to control group via Student’s t‐Test). (B-G) PLS-DA score plots of metabolomics data and pathway analysis in TCS-treated cells.

680

The PLS-DA parameters in (B), (C), (E), and (F) were based on two principal components (PCs) of the control and exposure groups.


Page 35 of 38


681 682

Figure 2. Changes of identified metabolite biomarkers in response to TCS exposure in the pathway of alanine, aspartate and glutamate metabolism

683

and purine metabolism. Data are the mean ± SD of n = 10, * P < 0.05, ** P < 0.01 (in comparison to control group via Student’s t‐Test); for XOD

684

activity, n = 4.



(A) L02

Control

TCS-1μM

TCS-2.5μM

(B) HepG2

Page 36 of 38

Control

TCS-1μM

TCS-10μM

685 686

Figure 3. Heatmap analysis of the identified lipid biomarkers in (A) L02 cells, and (B) HepG2 cells. The values were based on FCs of the lipid

687

peak intensity in comparison of the control.



2.5

FC (Lactate)

ADP

Amino acid catabolism







2.5

1.0 0.5 0.0

ATP

Amino Acids



1.5

0

0

1M 2.5M

L02

Pyruvate 

1M 10M

HepG2

Lactate CoA-SH

CoA-SH



2.5 2.0

0

1M 2.5M

0

L02

3.5 2.5 1.5

3.5

α-ketoglutarate

Fumarate



1.0

1M 10M

HepG2

NADH





0

1M 2.5M

NAD+

TCA Circle



0

Malate

HepG2

2.0



1.0

Isocitrate

Control TCS-Low TCS-High

3.0


3.0 2.0 1.5 1.0 0.5 0.0

0

1M 10M

HepG2

NAD+ + CoA-SH

2.5 2.0

0.5 1M 2.5M

0

L02

2.5 2.0

FC (FAD)

α-ketoglutarate Succinyl-CoA Fumarate Oxaloacetate

NADH + CO2

1M 10M

HepG2

FADH2

Succinyl-CoA FAD


2.0

GDP + Pi



1.5

3.0

1.0

2.5

0.5 0.0

Succinate

0

1M 2.5M

L02


2.0

GTP + CoA-SH 





1.5 1.0 0.5 0.0



1.5

0

1M 2.5M

L02

1.0 0.5 0.0

1.5

0

1M 2.5M

L02

1.0 0.5 0.0

2.5

FC (NADH)

0

FC (Succinate)

0.0



2.5

+

FC (Fumarate)

1M 10M



1.5

0.5 0.0



2.0

NAD+

1.0

1M 10M

HepG2

L02

NADH



0

1M 2.5M

0

2.5

0.0





1.5

0.0

FC (NAD )

FC (Malate)

3.0

0.5

0.5

Oxaloacetate







3.0

FC (Citrate)

Acetyl-CoA

3.5

Citrate 3.5



1.0

Pantothenic Acid

NADH + CO2

Fatty Acids

1.5

L02

NAD+ +

Fatty acid β-oxidation


2.0

FC (α -ketoglutarate)

Glucose


2.0

Glycolysis

FC (Pantothenic acid)

Page 37 of 38

Q

0

1M 2.5M

L02

C

Electron transport chain

ROS ATP

688

mitochondrial inner membrane

Oxidative Phosphorylation

689

Figure 4. Disturbance of energy metabolism in liver cells after exposure to TCS. Data

690

are the mean ± SD of n = 10, * P < 0.05, ** P < 0.01 (in comparison to control group

691

via Student’s t‐Test).



692 693

Figure 5. TCS-induced oxidative damage and anti-oxidative responses in liver cells. (A) ROS generation, (B) MDA content, (C) SOD activity,

694

(D) CAT activity, (E) GSH level, and (F) taurine level. Data are the mean ± SD of n = 6 (A), n= 4 (B, C, D) and n = 10 (E, F), * P < 0.05, ** P

Integration of Metabolomics and Lipidomics Reveals Metabolic

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