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Gene expression profiles and molecular mechanism of cultured human chondrocytes' exposure to T-2 toxin and deoxynivalenol
Accepted Manuscript Gene expression profiles and molecular mechanism of cultured human chondrocytes' exposure to T-2 toxin and deoxynivalenol Lei Yang, Jianping Zhang, Guanghui Zhao, Cuiyan Wu, Yujie Ning, Xiong Guo, Xi Wang, Mikko J. Lammi PII:
S0041-0101(17)30203-9
DOI:
10.1016/j.toxicon.2017.06.014
Reference:
TOXCON 5657
To appear in:
Toxicon
Received Date: 13 March 2017 Revised Date:
21 June 2017
Accepted Date: 28 June 2017
Please cite this article as: Yang, L., Zhang, J., Zhao, G., Wu, C., Ning, Y., Guo, X., Wang, X., Lammi, M.J., Gene expression profiles and molecular mechanism of cultured human chondrocytes' exposure to T-2 toxin and deoxynivalenol, Toxicon (2017), doi: 10.1016/j.toxicon.2017.06.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Gene expression profiles and molecular mechanism of cultured human
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chondrocytes’ exposure to T-2 toxin and deoxynivalenol
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Lei Yanga, Jianping Zhangb, Guanghui Zhaoc, Cuiyan Wua, Yujie Ninga, Xiong Guoa，*,
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Xi Wanga,*, Mikko J. Lammia,d
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a
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Laboratory of Trace Elements and Endemic Diseases of National Health and Family
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Planning Commission, Xi’an 710061, Shaanxi, PR of China
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School of Public Health, Health Science Center, Xi’an Jiaotong University, Key
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PR of China
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Shaanxi, PR of China
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d
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*Corresponding author.
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E-mail address: [email protected] (X. Guo), [email protected] (X.
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Wang).
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Hong Hui Hospital, Health Science Center, Xi’an Jiaotong University, Xi’an 710054,
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Department of Integrative Medical Biology, University of Umeå, Umeå, Sweden
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School of Clinical Medicine, Hainan Medical University, Haikou 571199, Hainan,
ACCEPTED MANUSCRIPT Abstract
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T-2 toxin and deoxynivalenol (DON) are secondary metabolites produced by
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Fusarium fungi and are commonly found on food and feed. Although T-2 toxin and
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DON have been suggested as the etiology of Kashin-Beck disease (KBD), an endemic
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osteochondropathy, little is known about the mechanism when human chondrocytes
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are exposed to T-2 toxin and DON. The purpose of this study is to identify the gene
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expression differences and underlying molecular changes modulated by T-2 toxin and
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DON in vitro in human chondrocytes. After the experiments of cell viability, the gene
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expression profiles were analyzed in cells that were treated with 0.01 µg /ml T-2 toxin
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and 1.0 µg /ml DON for 72 h by Affymetrix Human Gene Chip. The array results
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showed that 882 and 2118 genes were differentially expressed for T-2 toxin and DON
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exposure, respectively. Enrichment analysis revealed that diverse cellular processes
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including DNA damage, cell cycle regulation and metabolism of extracellular matrix
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were affected when human chondrocytes were exposed to T-2 toxin and DON. These
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results demonstrate the gene expression differences and molecular mechanism of
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cultured human chondrocytes exposure to T-2 toxin and DON, and provide a new
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insight into future research in the etiology of KBD.
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Keywords: T-2 toxin; Deoxynivalenol; Human chondrocytes; Microarray
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1. Introduction The trichothecenes are a large family of secondary metabolites produced by
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Fusarium fungi and are commonly found on food and feed (Marin et al., 2013).
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Chronic dietary exposure to trichothecenes results in toxic symptoms such as feed
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refusal, weight loss, diarrhea, neuroendocrine changes, immunological and
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hematological disorders (Rocha et al., 2005; Yang et al., 2015). T-2 toxin and
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deoxynivalenol (DON), belonging to type A and type B of the trichothecenes, have
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drawn much attention due to their frequency of occurrence and high toxicity (Bennett
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and Klich, 2003). T-2 toxin and DON are commonly found on cereals, such as maize,
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wheat, barley, oats and mixed feed (Desjardins et al., 1993; Wu et al., 2013). These
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two types of mycotoxins represent a health threat to humans and animals. Exposure to
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T-2 toxin and DON can cause lesions in the brain, hematopoietic, lymphoid,
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gastrointestinal tissues, and reducing reproductive organ functions (Rotter et al., 1996;
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Wu et al., 2010).
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Contamination of food by mycotoxins has been suggested to explain the aetiology
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of Kashin-Beck disease (KBD), which is a chronic and endemic osteochondropathy
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mainly found in China and other parts of Asia (Haubruge et al., 2001). T-2 toxin was
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the prominent suggested mycotoxins that contributed to KBD occurrence based on the
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epidemiologic studies in past decades (Yang, 1995), but a recent study also indicated
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that DON contamination in wheat flour was consistent with KBD prevalence (Lei et
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al., 2016). T-2 toxin and DON could induce apoptosis, mitochondria dysfunction and
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However, the molecular mechanism in detail of T-2 toxin and DON induced human
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chondrocytes, and its relationship with the pathological characteristics of
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chondrocytes in KBD were still unclear.
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The application of transcriptome microarray technology to the field of toxicology
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has provided the ability to determine changes in the expression of a large number of
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genes simultaneously, thereby obtaining information on molecular mechanism in
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greater detail following exposure to a toxic compound (Afshari et al., 1999). Several
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studies have provided the gene expression signatures induced by T-2 toxin or DON in
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vivo (Sehata et al., 2005; Sehata et al., 2004) and in vitro (Li et al., 2013). However,
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there is no gene expression data in the literature on human chondrocytes induced by
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T-2 toxin and DON to our knowledge. Therefore, the present study aimed to
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investigate the gene expression profiles and molecular mechanism of cultured human
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chondrocytes exposed to T-2 toxin and DON. This knowledge could contribute to a
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better understanding of the link between mycotoxins exposure and the development of
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human cartilage disease, such as KBD.
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2. Materials and methods
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2.1. Chemicals
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T-2 toxin solution (NO.34071, 100 µg/ml), DON solution (NO.34124, 100 µg/ml)
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and 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were
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eagle medium-F12 (DMEM-F12), fetal bovine serum (FBS), and penicillin/
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streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other
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chemicals used were of analytical grade.
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2.2. Specimens collection
Specimens of normal human articular cartilage were obtained from four donors
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(three males and a female, aged 48.2 ± 4.6 years old) who had suffered accident and
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subsequent amputation. All of the subjects were excluded osteoarthritis, rheumatoid
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arthritis and the genetic bone or cartilage diseases. All donors signed an informed
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consent. The study was approved by the Human Ethics Committee, Xi’an Jiaotong
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University.
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2.3. Cell culture
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Specimens were transported immediately in specimen bag with DMEM-F12 after
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surgery. After rinsing with phosphate buffered saline (PBS) three times, the articular
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cartilage were resected from bones and cut in to 1 mm3 slices in PBS with antibiotics
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(penicillin and streptomycin). Then the cartilage slices were incubated with 0.25%
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trypsin at room temperature for 30 min. After removing the trypsin solution, the
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cartilage slices were treated with 0.2% type II collagenase at 37ºC for 12~16 h.
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Chondrocytes were collected through gauze to filter undigested fragments and
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cultured in DMEM-F12 containing 10% FBS, 100 IU/mL penicillin and 100 µg/mL
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streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air. The first passage
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of cells was used in this study to ensure sufficient cells in all experiments. The
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concentrations of T-2 toxin and DON were made by direct dilution in culture medium.
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2.4. Cell viability assay
The determination of cell viability was measured by MTT assay. Briefly, cells
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were cultured in 96-well plates at 5×103 cells/well at 37 °C in a humidified atmosphere
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of 5% CO2 in air for 48 h until confluence. Then cells were treated with T-2 toxin (at
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0.001, 0.005, 0.01, 0.02 and 0.05 µg/mL), and DON (at 0.1, 0.5, 1.0, 2.0 and 5.0
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µg/mL) for 24, 48 and 72 h, respectively. After mycotoxins exposure, the medium was
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replaced by fresh medium containing 20 µl MTT solution. After 4 h incubation at
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37°C in humidified 5% CO2 atmosphere at dark, the medium was removed and
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replaced with 150 µl dimethyl sulfoxide (DMSO) to dissolve the formazan formed by
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metabolically active cells (van Meerloo et al., 2011). Then, plates were gentle shaking
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for 10 min. Measurement of the absorbance was performed with an automatic ELISA
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reader (Infinite M200, Tecan, Switzerland) at 490 nm with DMSO measured as a
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blank. All measurements were done in triplicate with five replicates per treatment.
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2.5. Experimental design
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The concentrations of T-2 toxin at 0.01 µg /ml and DON at 1.0 µg /ml were
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selected for further study. Cells were cultured in 25 cm2 culture flasks until 80%
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confluence. Then cells were treated with culture medium containing T-2 toxin and
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DON mentioned above for 72 h, respectively. Cells cultured with the same medium
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without mycotoxins as the controls.
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2.6. Total RNA extraction Total RNA was extracted separately with an E.Z.N.A.
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Total RNA Kit I from
Omega Bio-Tek, Inc. (R6834-02, Norcross, GA, USA) according to the
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manufacturer's protocol. RNA quantity and purity was verified by using a bioanalyser,
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and RNA integrity was determined by agarose gel electrophoresis. Only RNA with
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A260/A280 ≥ 1.8, and 28S/18S ratio larger than or close to 2 was considered suitable
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for further processing.
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2.7. Microarray hybridization and data analysis
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The gene expression profiles were carried out using GeneChip® PrimeView™
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Human Gene Expression Array (Affymetrix) according to the manufacturer’s
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instruction. Briefly, total RNA was first converted to double-stranded cDNA,
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followed by in vitro transcription to make cRNA. Then single-stranded cDNA was
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synthesized, end-labeled and hybridized to gene arrays. Both the RNA quality control
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tests and microarray analysis were conducted by CapitalBio Technology Inc.
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The data of GeneChip were analyzed using Affymetrix® GeneChip® Command
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Console® (AGCC) software. Data pre-processing including background correction
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and normalization used the RMA method (Irizarry et al., 2003). Differentially
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method by R software with the recruiting criteria of fold change ≥2 or ≤0.5, and
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q-value ≤ 5%. In addition, Gene Ontology (GO), and enriched pathways of the
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differentially expressed genes were identified using the KEGG Orthology Based
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Annotation System (KOBAS 2.0, http://kobas.cbi.pku.edu.cn/).
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2.8. Validation of gene expression by quantitative real-time PCR (qRT-PCR)
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We selected eight genes of interest (four up and four down-regulated), including
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CDKN1A, DDB2, GADD45A, CDC20, TIMP3, ACAN, CILP-2 and HAPLN1,
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which play important roles in cell cycle, DNA damage and metabolism in ECM, for
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qRT-PCR validation of the microarray data. The primers of selected genes
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(Supplemental Table S1) were designed and synthesized by Sangon Biotech Co. Ltd.
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(Shanghai, CHN). Total RNA was converted into cDNA using Prime Script RT Master
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Mix (Perfect Real Time) (RR036A, TAKARA). Briefly, 0.5 µg total RNA and 2 µL
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5×PrimeScript™ RT Master Mix were mixed on ice, and then RNase-free deionised
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water was added to 10 µL. The samples were incubated at 37°C for 15 min, at 85°C
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for 5 s and at 4°C hold. The qRT-PCR was performed using SYBR Premix Ex Taq II
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(Tli RNaseH Plus) (RR820A, TAKARA) with the Bio-Rad IQ5 PCR Detection
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System (Bio-Rad, Hercules, CA, USA). The reactions were set up in a total volume of
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25 µL using 12.5 µL SYBR Premix Ex Taq II, 1 µL PCR forward primer, 1 µL PCR
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reverse primer, 2 µL cDNA and 8.5 µL RNase –free deionized water. The cycles were
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set as 1 cycle of step 1 at 95 °C for 15 s, and 40 cycles of step 2 at 95 °C for 5 s and at
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60 °C for 30 s. Relative gene expression levels were normalized to GAPDH
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expression as it has been shown to be a relatively stable reference gene in cultured
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human articular chondrocytes (Ito et al., 2014).
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3. Results
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3.1. Cytotoxicity
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The effects of T-2 toxin and DON on proliferation of human chondrocytes after 24,
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48, and 72 h exposure were measured by MTT assay. Compared to the controls, T-2
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toxin (0.005 to 0.05 µg/ml) and DON (0.5 to 5.0 µg/ml) significantly reduced the cell
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viability of human chondrocytes (P<0.05). As expected, T-2 toxin (Fig. 1A) and DON
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(Fig.
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concentration-dependent manner. Based on these results, 0.01 µg/ml T-2 toxin and 1.0
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µg/ml DON treatment both decreased the viability of human chondrocytes nearly to
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60% for 72 h exposure, then we used 0.01 µg/ml T-2 toxin and 1.0 µg/ml DON after
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72 h exposure for the gene expression assay.
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3.2. Identification of differentially expressed genes In order to obtain a detailed molecular and cellular processes of human
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chondrocytes after T-2 toxin and DON exposure, we used a human genome
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microarray to investigate the differential gene expression profiles of human
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chondrocytes after treatment with 0.01 µg/ml T-2 toxin and 1.0 µg/ml DON for 72 h,
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respectively. The Human genome microarray analysis identified 882 genes (349 genes
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T-2 toxin exposure (Supplemental Table S2), while 2118 genes (1124 genes
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up-regulated and 994 genes down-regulated) were differentially expressed for DON
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exposure (Supplemental Table S3). In addition, 474 same genes (149 genes
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up-regulated and 325 genes down-regulated) were differentially expressed for both
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T-2 toxin and DON exposure. Only one dose of toxins was selected for this analysis,
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therefore, the data does not indicate whether dose-related responses.
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3.3. Gene ontology analysis
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The identification of enriched biological processes and molecular functions under
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GO category of human chondrocytes after T-2 toxin and DON exposure for 72 h was
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performed using KOBAS. The p-value < 0.05 denoted the significance of GO terms
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enrichment in the differentially expressed genes. For T-2 toxin exposure, a total of
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882 differentially expressed genes were enriched to 1228 significant GO terms of
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biological processes and 199 significant GO terms of molecular function
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(Supplemental Table S4). The top 10 significant enriched terms are shown in Fig. 2.
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The most prominent biological process involved in “histone H4-K20 demethylation”
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(p=2.64E-19), and the most prominent molecular function involved in ‘‘histone
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demethylase activity (H4-K20 specific)” (p= 2.64E-19). For DON exposure, a total of
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2118 differentially expressed genes were enriched to 1039 significant GO terms of
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biological processes and 155 significant GO terms of molecular function
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(Supplemental Table S5), the top 10 significant enriched terms shown in Fig. 3. The
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most prominent biological process involved in “mitotic cell cycle” (p= 7.47E-30), and
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the most prominent molecular function involved in “RNA binding” (p= 6.50E-32).
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3.4. Pathway analysis
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Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the
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altered pathways of human chondrocytes after T-2 toxin and DON treatment for 72 h
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were identified using KOBAS. The representative pathways (p value < 0.05) for T-2
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toxin and DON exposures are listed in Table 1 and Table 2, respectively. For T-2 toxin
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exposure, the differentially expressed genes involved in pathways such as viral
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carcinogenesis, p53 signaling pathway, systemic lupus erythematosus, alcoholism,
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lysosome, and protein processing. For DON exposure, the differentially expressed
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genes involved in pathways such as ECM-receptor interaction, cell cycle, RNA
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transport, protein digestion and absorption, spliceosome, and DNA replication. In
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addition, 14 pathways were significantly enriched both for T-2 toxin and DON
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exposure, such as ECM-receptor interaction, cell cycle, and PI3K-Akt signaling
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pathway.
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3.5. Genes validated by qRT-PCR Validation of the differential expression genes was done with qRT-PCR. We
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selected eight genes of interest including CDKN1A, DDB2, GADD45A, CDC20,
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TIMP3, ACAN, CILP-2 and HAPLN1. The results were expressed as fold change
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relative to control levels. As shown in Fig 4, the expression pattern showed good
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agreement with the microarray data, confirming the reliability of the microarray
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results.
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4. Discussion
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Although exposure to mycotoxins, such as T-2 toxin and DON, is suggested to
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associate with KBD, it has been difficult to establish a direct link between these
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toxins and KBD development. While numerous toxicological studies have focused on
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the adverse effects of T-2 toxin and DON on cartilage in vivo and in vitro (Chen et al.,
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2011; Debouck et al., 2001; Lu et al., 2012; Wang et al., 2011), the underlying
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molecular interactions, gene expression and the resulting secondary signaling
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responses of chondrocytes involved in these mycotoxins exposure are poorly known.
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In t This study we focuses on the effects of T-2 toxin and DON on gene expressions
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profiles and molecular mechanism of cultured human chondrocytes.
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Our toxicity study by MTT assay showed that T-2 toxin induced decrease of cell
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viability of human chondrocytes in a time- and concentration-dependent manner,
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which was consistent with the previous studies (Chen et al., 2011; Liu et al., 2014). In
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addition, many in vitro studies investigated the effect of DON on cell viability in
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different type of cells (Bensassi et al., 2009; Deng et al., 2016). A dose response
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relationship between DON and cell viability of human chondrocytes was initially
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investigated in our work, and the results We show here that DON significantly
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reduced cell viability at concentration of 0.5 to 5.0 µg/mL. We also found that the
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ACCEPTED MANUSCRIPT effective dose of T-2 toxin is nearly 100-times stronger than DON in human
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chondrocytes. Although all trichothecenes appear to inhibit peptidyl transferase by
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binding to the same ribosome-binding site, their different effects correlate with the
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different functional groups present in the toxins (Bennett and Klich, 2003). This may
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explain the big difference in the effective dose between T-2 toxin and DON, which
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belong to different types of nonmacrocylic trichothecenes.
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Previous studies have found that there were considerable genes and pathways
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involved in T-2 toxin and DON induced toxicity (Li et al., 2013; Sehata et al., 2005;
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Wentzel et al., 2016). In our study, normal human chondrocytes were treated with a
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single dose of T-2 toxin or DON for 72 h. Our results show This study indicates that
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the expressions of 349 genes were induced and 533 genes were suppressed by T-2
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toxin, while 1124 genes were induced and 994 genes were suppressed by DON.
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Although the gene expression profiles with T-2 toxin or DON treatment are distinct,
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there were 474 genes that showed similar tendency both with T-2 toxin and DON
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treatment. Moreover, the enrichment analysis of the differential expression genes in
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our study also provided a number of altered canonical pathways, which reflected the
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impact of T-2 toxin and DON on human chondrocytes. These results indicated that T-2
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toxin and DON seriously affected homeostasis of cultured human chondrocytes by
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altered gene expression.
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Since it is known that cell cycle arrest and DNA damage are vital toxicological
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surprising that several significantly altered genes in our microarray data are involved
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in cell cycle checkpoints, response to DNA damage and repair mechanism. The
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expressions of growth arrest and DNA damage inducible alpha (GADD45A) and
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damage specific DNA binding protein 2 (DDB2) in cultured human chondrocytes
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were both up-regulated with T-2 toxin and DON treatment. GADD45A is specifically
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involved in the repair of DNA by nucleotide excision and, thus, delays cell cycle
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progression when damage is detected (Hollander and Fornace, 2002). DDB2 is
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involved in early recognition of DNA damage and participates in nucleotide excision
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repair pathway (Fitch et al., 2003). In addition, the up-regulation of superoxide
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dismutase 2 (SOD2) indicated that oxidative DNA damage in human chondrocytes
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was induced by these two trichothecenes.
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Several other cell cycle progression related genes were also significantly altered
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both by T-2 toxin or DON, such as cyclin dependent kinase inhibitor 1A (CDKN1A),
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cyclin B1 (CCNB1) and cell division cycle 20 (CDC20), which were up-regulated,
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while cyclin D1 (CCND1) was down-regulated. Of those, CDKN1A (p21) is a critical
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intermediate as an inhibitor of cellular proliferation, and significant increases in p21
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mRNA by T-2 toxin and DON on murine macrophages RAW264.7 has been shown in
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a previous study (Wang et al., 2012). Cyclin D1 is regulator of CDK kinases and is
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essential for cell cycle G1/S transition (Walker and Assoian, 2005). However, our in
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vitro study found that T-2 toxin resulted in G0/G1 phase arrest, while DON resulted in
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G2/M phase arrest in human chondrocytic cell line (C28/I2) (data not shown). This
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suggests that cell cycle arrest induced by T-2 toxin and DON are different, but the
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mechanism is needed to be further explored.
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Many genes related to DNA replication were observed in our microarray data,
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which are also essential for S phase progression, The enrichment results refer to DNA
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replication given that altered genes were resulting in significantly enriched in GO
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terms of DNA replication. In addition, our pathway analysis also showed that some
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significantly altered KEGG pathways were related to cell cycle progression, such as
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cell cycle, p53 signaling pathway, PI3K-Akt signaling pathway and FoxO signaling
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pathway. Taken together, these findings suggest that T-2 toxin and DON are able to
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induce cell cycle arrest in response to DNA damage in human chondrocytes.
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As the main compositions of cartilage extracellular matrix (ECM), collagens and
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proteoglycans play important roles in cartilage function. The abnormal expression of
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type II collagen, aggrecan, and a family of proteolytic enzymes induced by T-2 toxin
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and DON in chondrocytes or engineered cartilage were observed in previous studies
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(Chen et al., 2011; Li et al., 2014; Li et al., 2008), which suggested a link between
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mycotoxins and cartilage degradation in KBD. The expression of ECM-related genes,
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such as extracellular matrix proteins (ECM1, ECM2), aggrecan (ACAN), collagens
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(COL1A2, COL9A1, COL11A1), tissue inhibitor of metalloproteinase-3 (TIMP3) and
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cartilage intermediate layer protein 2 (CILP2), were significantly down-regulated in
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CILP2 is a glycoprotein playing a role in cartilage scaffolding, and loss of CILP-2
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may involve in the pathophysiology of osteoarthritis (Bernardo et al., 2011).
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Moreover, our enrichment analysis revealed refer to ECM, showing that altered genes
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were significantly enriched in altered GO terms of ECM structure and organization,
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and enriched in the KEGG pathway of ECM-receptor interaction in the KEGG. These
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results suggest that toxic effect of T-2 toxin and DON on human chondrocytes involve
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in the imbalance of collagen and proteoglycan metabolism in ECM and contribute to
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cartilage degradation.
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The significantly enriched GO terms of histone demethylase were observed in our
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study, which indicated that potential epigenetic changes in human chondrocytes were
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induced by T-2 toxin and DON. Histone lysine methylation has been linked to the
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process of DNA repair (Botuyan et al., 2006), but understanding the mechanism of
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histone methylation in response to damage of trichothecenes in chondrocytes needs
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further study. Based on KEGG pathway results, the alteration of the main metabolic
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processes like protein biosynthesis or glycolysis and lysosome were observed in
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chondrocytes for T-2 and DON exposure. Additionally, other important pathways
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were also found to be altered in this study, such as Lysosome, Viral carcinogenesis,
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and Systemic lupus erythematosus. However, as the molecular mechanism of T-2
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toxin and DON toxicity effects is very complicated, it is difficult to explain the roles
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these pathways may play in toxicity of T-2 toxin and DON on human chondrocytes.
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5. Conclusion In summary, our results confirmed that T-2 toxin and DON significantly inhibit
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proliferation of chondrocytes. The microarray data provided the gene expression
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differences and molecular mechanism of chondrocytes after T-2 toxin and DON
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exposure. It shows that the toxicity effect of T-2 toxin and DON in human
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chondrocytes might involve in DNA damage, cell cycle regulation, metabolism of
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ECM and other biological process through specific signaling pathways. These
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observations provide new insight into our future research on the linkage between
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these mycotoxins and risk of KBD.
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Conflict of interest
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The authors have no conflict of interest.
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Acknowledgments
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This work was supported by the National Natural Scientific Foundation of China (No.
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81472924 and 81620108026).
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trichothecenes as DON and T-2 toxin. Toxicol. Sci. 127, 412-424.
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Wu, Q., Dohnal V., Huang L., Kuca, K., Yuan Z. 2010. Metabolic pathways of
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thichothecenes. Drug Metab. Rev. 42, 250-267. Wu, Q., Dohnal, V., Kuca, K., Yuan, Z., 2013. Trichothecenes: structure-toxic activity relationships. Curr. Drug Metab. 14, 641-660. Wu, Q.H., Wang, X., Yang, W., Nussler, A.K., Xiong, L.Y., Kuca, K., Dohnal, V.,
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Zhang, X.J., Yuan, Z.H., 2014. Oxidative stress-mediated cytotoxicity and
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Arch. Toxicol. 88, 1309-1326.
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Yang, J.B., 1995. Research report on the etiology of Kashin-Beck disease (KBD).
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Yang, S., Wang, Y., Beier, R.C., Zhang, H., De Ruyck, K., Sun, F., Cao, X., Shen, J.,
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Zhang, S., Wang, Z., 2015. Simultaneous determination of type A and B
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trichothecenes and their main metabolites in food animal tissues by
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ultraperformance liquid chromatography coupled with triple-quadrupole mass
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spectrometry. J. Agric. Food Chem. 63, 8592-8600.
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Figure legends
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Fig. 1. Cytotoxic effects of T-2 toxin and DON on human chondrocytes determined
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by MTT assay. (A-B) Cells were treated with T-2 toxin (at 0.001, 0.005, 0.01, 0.02
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and 0.05 µg/mL), and DON (at 0.1, 0.5, 1.0, 2.0 and 5.0 µg/mL) for 24, 48 and 72 h,
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respectively. Data are expressed as the means ± SD of three experiments. * represents
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p < 0.05 comparing with the control group (Student’s t-test).
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in human chondrocytes after T-2 toxin treatment.
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Fig. 3. Significant (top 10) GO terms of biological processes and molecular function
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in human chondrocytes after DON treatment.
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Fig. 4. The results of qRT-PCR validation experiment. (A) T-2 toxin treatment. (B)
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DON treatment. qRT-PCR results are represented as means ± SD.
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Supplementary Materials
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Table S1 Primers used for real-time quantitative PCR.
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Table S2 The complete list of differentially expressed genes in human chondrocytes
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after T-2 toxin treatment.
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Table S3 The complete list of differentially expressed genes in human chondrocytes
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after DON treatment.
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Table S4 Summary of the significant GO terms in human chondrocytes after T-2 toxin
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treatment.
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Table S5 Summary of the significant GO terms in human chondrocytes after DON
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treatment.
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p-value
27/205
6.79E-07
12/68
7.51E-05
17/135
0.000125
20/178
0.000137
15/122
0.000385
18/168
0.000488
8/44
0.000987
hsa04512: ECM-receptor interaction
11/87
0.00182
hsa05219: Bladder cancer
7/38
0.001898
hsa04974: Protein digestion and absorption
11/89
0.002141
hsa04110: Cell cycle
13/124
0.00335
hsa05134: Legionellosis
8/55
0.003458
hsa01230: Biosynthesis of amino acids
9/73
0.005337
hsa04151: PI3K-Akt signaling pathway
24/346
0.013936
4/20
0.014142
4/23
0.021263
hsa04115: p53 signaling pathway hsa05322: Systemic lupus erythematosus
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hsa05203: Viral carcinogenesis
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hsa04142: Lysosome
hsa04141: Protein processing in endoplasmic reticulum
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hsa00970: Aminoacyl-tRNA biosynthesis
hsa00532: Glycosaminoglycan biosynthesis-chondroitin sulfate/ dermatan sulfate hsa03060: Protein export
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0.021838
hsa00480: Glutathione metabolism
6/51
0.025583
hsa04068: FoxO signaling pathway
11/133
0.0296
hsa00750: Vitamin B6 metabolism
2/6
0.03793
hsa05206: MicroRNAs in cancer
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hsa00511: Other glycan degradation
7/72
0.038128
12/161
0.044902
3/18
0.049287
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hsa04918: Thyroid hormone synthesis
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hsa05144: Malaria
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p-value
28/87
1.26002E-08
hsa04110: Cell cycle
28/124
5.52287E-06
hsa03013: RNA transport
32/167
2.30663E-05
21/89
4.5679E-05
26/131
7.85738E-05
12/36
0.000138506
33/207
0.000378143
47/346
0.000683863
22/122
0.000840001
hsa00511: Other glycan degradation
7/18
0.001693268
hsa00480: Glutathione metabolism
12/51
0.001904167
hsa05203: Viral carcinogenesis
30/205
0.002227161
hsa04115: p53 signaling pathway
14/68
0.002471848
hsa05322: Systemic lupus erythematosus
22/135
0.0026108
hsa03008: Ribosome biogenesis in
15/77
0.0028106
7/23
0.005191215
25/178
0.007768235
hsa04974: Protein digestion and absorption hsa03040: Spliceosome hsa03030: DNA replication hsa04510: Focal adhesion
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hsa04151: PI3K-Akt signaling pathway
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hsa04512: ECM-receptor interaction
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eukaryotes hsa03430: Mismatch repair hsa05034: Alcoholism
10/47
0.008057865
hsa05144: Malaria
10/49
0.010266479
hsa05146: Amoebiasis
17/109
0.010886909
hsa05206: MicroRNAs in cancer
22/161
0.015528637
hsa04145: Phagosome
21/155
0.01916914
hsa03015: mRNA surveillance pathway
14/91
0.021545796
hsa04114: Oocyte meiosis
16/113
0.027331791
13/86
0.029216548
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hsa03420: Nucleotide excision repair
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hsa05222: Small cell lung cancer
10/62
0.037227114
hsa05166: HTLV-I infection
30/261
0.038304791
hsa01230: Biosynthesis of amino acids
11/73
0.04333637
hsa04068: FoxO signaling pathway
17/133
0.049423343
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hsa00590: Arachidonic acid metabolism
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ACCEPTED MANUSCRIPT 1. Microarray analysis of gene expression profiles of human chondrocytes exposed to T-2 toxin and DON was performed. 2. Bioinformatic analysis showed the potential molecular mechanism.
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3. DNA damage, cell cycle regulation and metabolism of extracellular matrix were
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mainly affected.
S0041-0101(17)30203-9
DOI:
10.1016/j.toxicon.2017.06.014
Reference:
TOXCON 5657
To appear in:
Toxicon
Received Date: 13 March 2017 Revised Date:
21 June 2017
Accepted Date: 28 June 2017
Please cite this article as: Yang, L., Zhang, J., Zhao, G., Wu, C., Ning, Y., Guo, X., Wang, X., Lammi, M.J., Gene expression profiles and molecular mechanism of cultured human chondrocytes' exposure to T-2 toxin and deoxynivalenol, Toxicon (2017), doi: 10.1016/j.toxicon.2017.06.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Gene expression profiles and molecular mechanism of cultured human
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chondrocytes’ exposure to T-2 toxin and deoxynivalenol
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Lei Yanga, Jianping Zhangb, Guanghui Zhaoc, Cuiyan Wua, Yujie Ninga, Xiong Guoa，*,
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Xi Wanga,*, Mikko J. Lammia,d
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a
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Laboratory of Trace Elements and Endemic Diseases of National Health and Family
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Planning Commission, Xi’an 710061, Shaanxi, PR of China
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School of Public Health, Health Science Center, Xi’an Jiaotong University, Key
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b
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PR of China
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c
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Shaanxi, PR of China
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d
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*Corresponding author.
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E-mail address: [email protected] (X. Guo), [email protected] (X.
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Wang).
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Hong Hui Hospital, Health Science Center, Xi’an Jiaotong University, Xi’an 710054,
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Department of Integrative Medical Biology, University of Umeå, Umeå, Sweden
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School of Clinical Medicine, Hainan Medical University, Haikou 571199, Hainan,
ACCEPTED MANUSCRIPT Abstract
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T-2 toxin and deoxynivalenol (DON) are secondary metabolites produced by
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Fusarium fungi and are commonly found on food and feed. Although T-2 toxin and
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DON have been suggested as the etiology of Kashin-Beck disease (KBD), an endemic
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osteochondropathy, little is known about the mechanism when human chondrocytes
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are exposed to T-2 toxin and DON. The purpose of this study is to identify the gene
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expression differences and underlying molecular changes modulated by T-2 toxin and
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DON in vitro in human chondrocytes. After the experiments of cell viability, the gene
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expression profiles were analyzed in cells that were treated with 0.01 µg /ml T-2 toxin
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and 1.0 µg /ml DON for 72 h by Affymetrix Human Gene Chip. The array results
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showed that 882 and 2118 genes were differentially expressed for T-2 toxin and DON
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exposure, respectively. Enrichment analysis revealed that diverse cellular processes
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including DNA damage, cell cycle regulation and metabolism of extracellular matrix
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were affected when human chondrocytes were exposed to T-2 toxin and DON. These
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results demonstrate the gene expression differences and molecular mechanism of
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cultured human chondrocytes exposure to T-2 toxin and DON, and provide a new
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insight into future research in the etiology of KBD.
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Keywords: T-2 toxin; Deoxynivalenol; Human chondrocytes; Microarray
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1. Introduction The trichothecenes are a large family of secondary metabolites produced by
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Fusarium fungi and are commonly found on food and feed (Marin et al., 2013).
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Chronic dietary exposure to trichothecenes results in toxic symptoms such as feed
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refusal, weight loss, diarrhea, neuroendocrine changes, immunological and
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hematological disorders (Rocha et al., 2005; Yang et al., 2015). T-2 toxin and
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deoxynivalenol (DON), belonging to type A and type B of the trichothecenes, have
46
drawn much attention due to their frequency of occurrence and high toxicity (Bennett
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and Klich, 2003). T-2 toxin and DON are commonly found on cereals, such as maize,
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wheat, barley, oats and mixed feed (Desjardins et al., 1993; Wu et al., 2013). These
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two types of mycotoxins represent a health threat to humans and animals. Exposure to
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T-2 toxin and DON can cause lesions in the brain, hematopoietic, lymphoid,
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gastrointestinal tissues, and reducing reproductive organ functions (Rotter et al., 1996;
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Wu et al., 2010).
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Contamination of food by mycotoxins has been suggested to explain the aetiology
55
of Kashin-Beck disease (KBD), which is a chronic and endemic osteochondropathy
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mainly found in China and other parts of Asia (Haubruge et al., 2001). T-2 toxin was
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the prominent suggested mycotoxins that contributed to KBD occurrence based on the
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epidemiologic studies in past decades (Yang, 1995), but a recent study also indicated
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that DON contamination in wheat flour was consistent with KBD prevalence (Lei et
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al., 2016). T-2 toxin and DON could induce apoptosis, mitochondria dysfunction and
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However, the molecular mechanism in detail of T-2 toxin and DON induced human
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chondrocytes, and its relationship with the pathological characteristics of
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chondrocytes in KBD were still unclear.
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The application of transcriptome microarray technology to the field of toxicology
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has provided the ability to determine changes in the expression of a large number of
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genes simultaneously, thereby obtaining information on molecular mechanism in
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greater detail following exposure to a toxic compound (Afshari et al., 1999). Several
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studies have provided the gene expression signatures induced by T-2 toxin or DON in
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vivo (Sehata et al., 2005; Sehata et al., 2004) and in vitro (Li et al., 2013). However,
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there is no gene expression data in the literature on human chondrocytes induced by
73
T-2 toxin and DON to our knowledge. Therefore, the present study aimed to
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investigate the gene expression profiles and molecular mechanism of cultured human
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chondrocytes exposed to T-2 toxin and DON. This knowledge could contribute to a
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better understanding of the link between mycotoxins exposure and the development of
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human cartilage disease, such as KBD.
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2. Materials and methods
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2.1. Chemicals
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T-2 toxin solution (NO.34071, 100 µg/ml), DON solution (NO.34124, 100 µg/ml)
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and 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were
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eagle medium-F12 (DMEM-F12), fetal bovine serum (FBS), and penicillin/
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streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other
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chemicals used were of analytical grade.
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2.2. Specimens collection
Specimens of normal human articular cartilage were obtained from four donors
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(three males and a female, aged 48.2 ± 4.6 years old) who had suffered accident and
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subsequent amputation. All of the subjects were excluded osteoarthritis, rheumatoid
92
arthritis and the genetic bone or cartilage diseases. All donors signed an informed
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consent. The study was approved by the Human Ethics Committee, Xi’an Jiaotong
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University.
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2.3. Cell culture
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Specimens were transported immediately in specimen bag with DMEM-F12 after
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surgery. After rinsing with phosphate buffered saline (PBS) three times, the articular
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cartilage were resected from bones and cut in to 1 mm3 slices in PBS with antibiotics
100
(penicillin and streptomycin). Then the cartilage slices were incubated with 0.25%
101
trypsin at room temperature for 30 min. After removing the trypsin solution, the
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cartilage slices were treated with 0.2% type II collagenase at 37ºC for 12~16 h.
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Chondrocytes were collected through gauze to filter undigested fragments and
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cultured in DMEM-F12 containing 10% FBS, 100 IU/mL penicillin and 100 µg/mL
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streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air. The first passage
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of cells was used in this study to ensure sufficient cells in all experiments. The
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concentrations of T-2 toxin and DON were made by direct dilution in culture medium.
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2.4. Cell viability assay
The determination of cell viability was measured by MTT assay. Briefly, cells
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were cultured in 96-well plates at 5×103 cells/well at 37 °C in a humidified atmosphere
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of 5% CO2 in air for 48 h until confluence. Then cells were treated with T-2 toxin (at
113
0.001, 0.005, 0.01, 0.02 and 0.05 µg/mL), and DON (at 0.1, 0.5, 1.0, 2.0 and 5.0
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µg/mL) for 24, 48 and 72 h, respectively. After mycotoxins exposure, the medium was
115
replaced by fresh medium containing 20 µl MTT solution. After 4 h incubation at
116
37°C in humidified 5% CO2 atmosphere at dark, the medium was removed and
117
replaced with 150 µl dimethyl sulfoxide (DMSO) to dissolve the formazan formed by
118
metabolically active cells (van Meerloo et al., 2011). Then, plates were gentle shaking
119
for 10 min. Measurement of the absorbance was performed with an automatic ELISA
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reader (Infinite M200, Tecan, Switzerland) at 490 nm with DMSO measured as a
121
blank. All measurements were done in triplicate with five replicates per treatment.
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2.5. Experimental design
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The concentrations of T-2 toxin at 0.01 µg /ml and DON at 1.0 µg /ml were
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selected for further study. Cells were cultured in 25 cm2 culture flasks until 80%
126
confluence. Then cells were treated with culture medium containing T-2 toxin and
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DON mentioned above for 72 h, respectively. Cells cultured with the same medium
128
without mycotoxins as the controls.
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2.6. Total RNA extraction Total RNA was extracted separately with an E.Z.N.A.
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®
Total RNA Kit I from
Omega Bio-Tek, Inc. (R6834-02, Norcross, GA, USA) according to the
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manufacturer's protocol. RNA quantity and purity was verified by using a bioanalyser,
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and RNA integrity was determined by agarose gel electrophoresis. Only RNA with
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A260/A280 ≥ 1.8, and 28S/18S ratio larger than or close to 2 was considered suitable
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for further processing.
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2.7. Microarray hybridization and data analysis
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The gene expression profiles were carried out using GeneChip® PrimeView™
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Human Gene Expression Array (Affymetrix) according to the manufacturer’s
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instruction. Briefly, total RNA was first converted to double-stranded cDNA,
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followed by in vitro transcription to make cRNA. Then single-stranded cDNA was
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synthesized, end-labeled and hybridized to gene arrays. Both the RNA quality control
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tests and microarray analysis were conducted by CapitalBio Technology Inc.
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The data of GeneChip were analyzed using Affymetrix® GeneChip® Command
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Console® (AGCC) software. Data pre-processing including background correction
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and normalization used the RMA method (Irizarry et al., 2003). Differentially
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method by R software with the recruiting criteria of fold change ≥2 or ≤0.5, and
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q-value ≤ 5%. In addition, Gene Ontology (GO), and enriched pathways of the
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differentially expressed genes were identified using the KEGG Orthology Based
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Annotation System (KOBAS 2.0, http://kobas.cbi.pku.edu.cn/).
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2.8. Validation of gene expression by quantitative real-time PCR (qRT-PCR)
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We selected eight genes of interest (four up and four down-regulated), including
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CDKN1A, DDB2, GADD45A, CDC20, TIMP3, ACAN, CILP-2 and HAPLN1,
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which play important roles in cell cycle, DNA damage and metabolism in ECM, for
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qRT-PCR validation of the microarray data. The primers of selected genes
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(Supplemental Table S1) were designed and synthesized by Sangon Biotech Co. Ltd.
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(Shanghai, CHN). Total RNA was converted into cDNA using Prime Script RT Master
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Mix (Perfect Real Time) (RR036A, TAKARA). Briefly, 0.5 µg total RNA and 2 µL
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5×PrimeScript™ RT Master Mix were mixed on ice, and then RNase-free deionised
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water was added to 10 µL. The samples were incubated at 37°C for 15 min, at 85°C
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for 5 s and at 4°C hold. The qRT-PCR was performed using SYBR Premix Ex Taq II
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(Tli RNaseH Plus) (RR820A, TAKARA) with the Bio-Rad IQ5 PCR Detection
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System (Bio-Rad, Hercules, CA, USA). The reactions were set up in a total volume of
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25 µL using 12.5 µL SYBR Premix Ex Taq II, 1 µL PCR forward primer, 1 µL PCR
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reverse primer, 2 µL cDNA and 8.5 µL RNase –free deionized water. The cycles were
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set as 1 cycle of step 1 at 95 °C for 15 s, and 40 cycles of step 2 at 95 °C for 5 s and at
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60 °C for 30 s. Relative gene expression levels were normalized to GAPDH
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expression as it has been shown to be a relatively stable reference gene in cultured
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human articular chondrocytes (Ito et al., 2014).
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3. Results
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3.1. Cytotoxicity
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The effects of T-2 toxin and DON on proliferation of human chondrocytes after 24,
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48, and 72 h exposure were measured by MTT assay. Compared to the controls, T-2
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toxin (0.005 to 0.05 µg/ml) and DON (0.5 to 5.0 µg/ml) significantly reduced the cell
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viability of human chondrocytes (P<0.05). As expected, T-2 toxin (Fig. 1A) and DON
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(Fig.
182
concentration-dependent manner. Based on these results, 0.01 µg/ml T-2 toxin and 1.0
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µg/ml DON treatment both decreased the viability of human chondrocytes nearly to
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60% for 72 h exposure, then we used 0.01 µg/ml T-2 toxin and 1.0 µg/ml DON after
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72 h exposure for the gene expression assay.
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3.2. Identification of differentially expressed genes In order to obtain a detailed molecular and cellular processes of human
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chondrocytes after T-2 toxin and DON exposure, we used a human genome
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microarray to investigate the differential gene expression profiles of human
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chondrocytes after treatment with 0.01 µg/ml T-2 toxin and 1.0 µg/ml DON for 72 h,
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respectively. The Human genome microarray analysis identified 882 genes (349 genes
ACCEPTED MANUSCRIPT up-regulated and 533 genes down-regulated), which were differentially expressed for
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T-2 toxin exposure (Supplemental Table S2), while 2118 genes (1124 genes
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up-regulated and 994 genes down-regulated) were differentially expressed for DON
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exposure (Supplemental Table S3). In addition, 474 same genes (149 genes
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up-regulated and 325 genes down-regulated) were differentially expressed for both
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T-2 toxin and DON exposure. Only one dose of toxins was selected for this analysis,
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therefore, the data does not indicate whether dose-related responses.
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3.3. Gene ontology analysis
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The identification of enriched biological processes and molecular functions under
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GO category of human chondrocytes after T-2 toxin and DON exposure for 72 h was
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performed using KOBAS. The p-value < 0.05 denoted the significance of GO terms
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enrichment in the differentially expressed genes. For T-2 toxin exposure, a total of
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882 differentially expressed genes were enriched to 1228 significant GO terms of
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biological processes and 199 significant GO terms of molecular function
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(Supplemental Table S4). The top 10 significant enriched terms are shown in Fig. 2.
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The most prominent biological process involved in “histone H4-K20 demethylation”
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(p=2.64E-19), and the most prominent molecular function involved in ‘‘histone
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demethylase activity (H4-K20 specific)” (p= 2.64E-19). For DON exposure, a total of
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2118 differentially expressed genes were enriched to 1039 significant GO terms of
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biological processes and 155 significant GO terms of molecular function
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(Supplemental Table S5), the top 10 significant enriched terms shown in Fig. 3. The
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most prominent biological process involved in “mitotic cell cycle” (p= 7.47E-30), and
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the most prominent molecular function involved in “RNA binding” (p= 6.50E-32).
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3.4. Pathway analysis
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Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the
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altered pathways of human chondrocytes after T-2 toxin and DON treatment for 72 h
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were identified using KOBAS. The representative pathways (p value < 0.05) for T-2
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toxin and DON exposures are listed in Table 1 and Table 2, respectively. For T-2 toxin
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exposure, the differentially expressed genes involved in pathways such as viral
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carcinogenesis, p53 signaling pathway, systemic lupus erythematosus, alcoholism,
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lysosome, and protein processing. For DON exposure, the differentially expressed
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genes involved in pathways such as ECM-receptor interaction, cell cycle, RNA
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transport, protein digestion and absorption, spliceosome, and DNA replication. In
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addition, 14 pathways were significantly enriched both for T-2 toxin and DON
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exposure, such as ECM-receptor interaction, cell cycle, and PI3K-Akt signaling
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pathway.
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3.5. Genes validated by qRT-PCR Validation of the differential expression genes was done with qRT-PCR. We
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selected eight genes of interest including CDKN1A, DDB2, GADD45A, CDC20,
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TIMP3, ACAN, CILP-2 and HAPLN1. The results were expressed as fold change
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relative to control levels. As shown in Fig 4, the expression pattern showed good
ACCEPTED MANUSCRIPT 237
agreement with the microarray data, confirming the reliability of the microarray
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results.
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4. Discussion
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Although exposure to mycotoxins, such as T-2 toxin and DON, is suggested to
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associate with KBD, it has been difficult to establish a direct link between these
243
toxins and KBD development. While numerous toxicological studies have focused on
244
the adverse effects of T-2 toxin and DON on cartilage in vivo and in vitro (Chen et al.,
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2011; Debouck et al., 2001; Lu et al., 2012; Wang et al., 2011), the underlying
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molecular interactions, gene expression and the resulting secondary signaling
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responses of chondrocytes involved in these mycotoxins exposure are poorly known.
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In t This study we focuses on the effects of T-2 toxin and DON on gene expressions
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profiles and molecular mechanism of cultured human chondrocytes.
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Our toxicity study by MTT assay showed that T-2 toxin induced decrease of cell
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viability of human chondrocytes in a time- and concentration-dependent manner,
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which was consistent with the previous studies (Chen et al., 2011; Liu et al., 2014). In
254
addition, many in vitro studies investigated the effect of DON on cell viability in
255
different type of cells (Bensassi et al., 2009; Deng et al., 2016). A dose response
256
relationship between DON and cell viability of human chondrocytes was initially
257
investigated in our work, and the results We show here that DON significantly
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reduced cell viability at concentration of 0.5 to 5.0 µg/mL. We also found that the
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ACCEPTED MANUSCRIPT effective dose of T-2 toxin is nearly 100-times stronger than DON in human
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chondrocytes. Although all trichothecenes appear to inhibit peptidyl transferase by
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binding to the same ribosome-binding site, their different effects correlate with the
262
different functional groups present in the toxins (Bennett and Klich, 2003). This may
263
explain the big difference in the effective dose between T-2 toxin and DON, which
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belong to different types of nonmacrocylic trichothecenes.
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Previous studies have found that there were considerable genes and pathways
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involved in T-2 toxin and DON induced toxicity (Li et al., 2013; Sehata et al., 2005;
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Wentzel et al., 2016). In our study, normal human chondrocytes were treated with a
269
single dose of T-2 toxin or DON for 72 h. Our results show This study indicates that
270
the expressions of 349 genes were induced and 533 genes were suppressed by T-2
271
toxin, while 1124 genes were induced and 994 genes were suppressed by DON.
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Although the gene expression profiles with T-2 toxin or DON treatment are distinct,
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there were 474 genes that showed similar tendency both with T-2 toxin and DON
274
treatment. Moreover, the enrichment analysis of the differential expression genes in
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our study also provided a number of altered canonical pathways, which reflected the
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impact of T-2 toxin and DON on human chondrocytes. These results indicated that T-2
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toxin and DON seriously affected homeostasis of cultured human chondrocytes by
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altered gene expression.
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Since it is known that cell cycle arrest and DNA damage are vital toxicological
ACCEPTED MANUSCRIPT mechanisms of trichothecenes on cell proliferation (Wu et al., 2014), it is not
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surprising that several significantly altered genes in our microarray data are involved
283
in cell cycle checkpoints, response to DNA damage and repair mechanism. The
284
expressions of growth arrest and DNA damage inducible alpha (GADD45A) and
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damage specific DNA binding protein 2 (DDB2) in cultured human chondrocytes
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were both up-regulated with T-2 toxin and DON treatment. GADD45A is specifically
287
involved in the repair of DNA by nucleotide excision and, thus, delays cell cycle
288
progression when damage is detected (Hollander and Fornace, 2002). DDB2 is
289
involved in early recognition of DNA damage and participates in nucleotide excision
290
repair pathway (Fitch et al., 2003). In addition, the up-regulation of superoxide
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dismutase 2 (SOD2) indicated that oxidative DNA damage in human chondrocytes
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was induced by these two trichothecenes.
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Several other cell cycle progression related genes were also significantly altered
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both by T-2 toxin or DON, such as cyclin dependent kinase inhibitor 1A (CDKN1A),
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cyclin B1 (CCNB1) and cell division cycle 20 (CDC20), which were up-regulated,
297
while cyclin D1 (CCND1) was down-regulated. Of those, CDKN1A (p21) is a critical
298
intermediate as an inhibitor of cellular proliferation, and significant increases in p21
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mRNA by T-2 toxin and DON on murine macrophages RAW264.7 has been shown in
300
a previous study (Wang et al., 2012). Cyclin D1 is regulator of CDK kinases and is
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essential for cell cycle G1/S transition (Walker and Assoian, 2005). However, our in
302
vitro study found that T-2 toxin resulted in G0/G1 phase arrest, while DON resulted in
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G2/M phase arrest in human chondrocytic cell line (C28/I2) (data not shown). This
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suggests that cell cycle arrest induced by T-2 toxin and DON are different, but the
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mechanism is needed to be further explored.
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Many genes related to DNA replication were observed in our microarray data,
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which are also essential for S phase progression, The enrichment results refer to DNA
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replication given that altered genes were resulting in significantly enriched in GO
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terms of DNA replication. In addition, our pathway analysis also showed that some
311
significantly altered KEGG pathways were related to cell cycle progression, such as
312
cell cycle, p53 signaling pathway, PI3K-Akt signaling pathway and FoxO signaling
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pathway. Taken together, these findings suggest that T-2 toxin and DON are able to
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induce cell cycle arrest in response to DNA damage in human chondrocytes.
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As the main compositions of cartilage extracellular matrix (ECM), collagens and
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proteoglycans play important roles in cartilage function. The abnormal expression of
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type II collagen, aggrecan, and a family of proteolytic enzymes induced by T-2 toxin
319
and DON in chondrocytes or engineered cartilage were observed in previous studies
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(Chen et al., 2011; Li et al., 2014; Li et al., 2008), which suggested a link between
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mycotoxins and cartilage degradation in KBD. The expression of ECM-related genes,
322
such as extracellular matrix proteins (ECM1, ECM2), aggrecan (ACAN), collagens
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(COL1A2, COL9A1, COL11A1), tissue inhibitor of metalloproteinase-3 (TIMP3) and
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cartilage intermediate layer protein 2 (CILP2), were significantly down-regulated in
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ACCEPTED MANUSCRIPT human chondrocytes both by T-2 toxin and DON treatment in the present study.
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CILP2 is a glycoprotein playing a role in cartilage scaffolding, and loss of CILP-2
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may involve in the pathophysiology of osteoarthritis (Bernardo et al., 2011).
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Moreover, our enrichment analysis revealed refer to ECM, showing that altered genes
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were significantly enriched in altered GO terms of ECM structure and organization,
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and enriched in the KEGG pathway of ECM-receptor interaction in the KEGG. These
331
results suggest that toxic effect of T-2 toxin and DON on human chondrocytes involve
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in the imbalance of collagen and proteoglycan metabolism in ECM and contribute to
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cartilage degradation.
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The significantly enriched GO terms of histone demethylase were observed in our
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study, which indicated that potential epigenetic changes in human chondrocytes were
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induced by T-2 toxin and DON. Histone lysine methylation has been linked to the
338
process of DNA repair (Botuyan et al., 2006), but understanding the mechanism of
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histone methylation in response to damage of trichothecenes in chondrocytes needs
340
further study. Based on KEGG pathway results, the alteration of the main metabolic
341
processes like protein biosynthesis or glycolysis and lysosome were observed in
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chondrocytes for T-2 and DON exposure. Additionally, other important pathways
343
were also found to be altered in this study, such as Lysosome, Viral carcinogenesis,
344
and Systemic lupus erythematosus. However, as the molecular mechanism of T-2
345
toxin and DON toxicity effects is very complicated, it is difficult to explain the roles
346
these pathways may play in toxicity of T-2 toxin and DON on human chondrocytes.
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5. Conclusion In summary, our results confirmed that T-2 toxin and DON significantly inhibit
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proliferation of chondrocytes. The microarray data provided the gene expression
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differences and molecular mechanism of chondrocytes after T-2 toxin and DON
352
exposure. It shows that the toxicity effect of T-2 toxin and DON in human
353
chondrocytes might involve in DNA damage, cell cycle regulation, metabolism of
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ECM and other biological process through specific signaling pathways. These
355
observations provide new insight into our future research on the linkage between
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these mycotoxins and risk of KBD.
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Conflict of interest
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The authors have no conflict of interest.
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Acknowledgments
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This work was supported by the National Natural Scientific Foundation of China (No.
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81472924 and 81620108026).
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Figure legends
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Fig. 1. Cytotoxic effects of T-2 toxin and DON on human chondrocytes determined
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by MTT assay. (A-B) Cells were treated with T-2 toxin (at 0.001, 0.005, 0.01, 0.02
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and 0.05 µg/mL), and DON (at 0.1, 0.5, 1.0, 2.0 and 5.0 µg/mL) for 24, 48 and 72 h,
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respectively. Data are expressed as the means ± SD of three experiments. * represents
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p < 0.05 comparing with the control group (Student’s t-test).
ACCEPTED MANUSCRIPT Fig. 2. Significant (top 10) GO terms of biological processes and molecular function
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in human chondrocytes after T-2 toxin treatment.
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Fig. 3. Significant (top 10) GO terms of biological processes and molecular function
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in human chondrocytes after DON treatment.
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Fig. 4. The results of qRT-PCR validation experiment. (A) T-2 toxin treatment. (B)
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DON treatment. qRT-PCR results are represented as means ± SD.
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SC
485
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Supplementary Materials
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Table S1 Primers used for real-time quantitative PCR.
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Table S2 The complete list of differentially expressed genes in human chondrocytes
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after T-2 toxin treatment.
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Table S3 The complete list of differentially expressed genes in human chondrocytes
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after DON treatment.
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Table S4 Summary of the significant GO terms in human chondrocytes after T-2 toxin
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treatment.
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Table S5 Summary of the significant GO terms in human chondrocytes after DON
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treatment.
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ACCEPTED MANUSCRIPT Table 1 Significant enriched KEGG pathways in human chondrocytes after T-2 toxin treatment No. of modulated KEGG category
p-value
27/205
6.79E-07
12/68
7.51E-05
17/135
0.000125
20/178
0.000137
15/122
0.000385
18/168
0.000488
8/44
0.000987
hsa04512: ECM-receptor interaction
11/87
0.00182
hsa05219: Bladder cancer
7/38
0.001898
hsa04974: Protein digestion and absorption
11/89
0.002141
hsa04110: Cell cycle
13/124
0.00335
hsa05134: Legionellosis
8/55
0.003458
hsa01230: Biosynthesis of amino acids
9/73
0.005337
hsa04151: PI3K-Akt signaling pathway
24/346
0.013936
4/20
0.014142
4/23
0.021263
hsa04115: p53 signaling pathway hsa05322: Systemic lupus erythematosus
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hsa05034: Alcoholism
SC
hsa05203: Viral carcinogenesis
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genes
hsa04142: Lysosome
hsa04141: Protein processing in endoplasmic reticulum
AC C
EP
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hsa00970: Aminoacyl-tRNA biosynthesis
hsa00532: Glycosaminoglycan biosynthesis-chondroitin sulfate/ dermatan sulfate hsa03060: Protein export
ACCEPTED MANUSCRIPT 6/49
0.021838
hsa00480: Glutathione metabolism
6/51
0.025583
hsa04068: FoxO signaling pathway
11/133
0.0296
hsa00750: Vitamin B6 metabolism
2/6
0.03793
hsa05206: MicroRNAs in cancer
AC C
EP
TE D
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hsa00511: Other glycan degradation
7/72
0.038128
12/161
0.044902
3/18
0.049287
SC
hsa04918: Thyroid hormone synthesis
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hsa05144: Malaria
ACCEPTED MANUSCRIPT Table 2 Significant enriched KEGG pathways in human chondrocytes after DON treatment No. of modulated KEGG category
p-value
28/87
1.26002E-08
hsa04110: Cell cycle
28/124
5.52287E-06
hsa03013: RNA transport
32/167
2.30663E-05
21/89
4.5679E-05
26/131
7.85738E-05
12/36
0.000138506
33/207
0.000378143
47/346
0.000683863
22/122
0.000840001
hsa00511: Other glycan degradation
7/18
0.001693268
hsa00480: Glutathione metabolism
12/51
0.001904167
hsa05203: Viral carcinogenesis
30/205
0.002227161
hsa04115: p53 signaling pathway
14/68
0.002471848
hsa05322: Systemic lupus erythematosus
22/135
0.0026108
hsa03008: Ribosome biogenesis in
15/77
0.0028106
7/23
0.005191215
25/178
0.007768235
hsa04974: Protein digestion and absorption hsa03040: Spliceosome hsa03030: DNA replication hsa04510: Focal adhesion
AC C
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hsa04142: Lysosome
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hsa04151: PI3K-Akt signaling pathway
SC
hsa04512: ECM-receptor interaction
M AN U
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genes
eukaryotes hsa03430: Mismatch repair hsa05034: Alcoholism
10/47
0.008057865
hsa05144: Malaria
10/49
0.010266479
hsa05146: Amoebiasis
17/109
0.010886909
hsa05206: MicroRNAs in cancer
22/161
0.015528637
hsa04145: Phagosome
21/155
0.01916914
hsa03015: mRNA surveillance pathway
14/91
0.021545796
hsa04114: Oocyte meiosis
16/113
0.027331791
13/86
0.029216548
SC
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hsa03420: Nucleotide excision repair
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hsa05222: Small cell lung cancer
10/62
0.037227114
hsa05166: HTLV-I infection
30/261
0.038304791
hsa01230: Biosynthesis of amino acids
11/73
0.04333637
hsa04068: FoxO signaling pathway
17/133
0.049423343
AC C
EP
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hsa00590: Arachidonic acid metabolism
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EP
TE D
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SC
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SC
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AC C
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TE D
M AN U
SC
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT 1. Microarray analysis of gene expression profiles of human chondrocytes exposed to T-2 toxin and DON was performed. 2. Bioinformatic analysis showed the potential molecular mechanism.
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3. DNA damage, cell cycle regulation and metabolism of extracellular matrix were
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mainly affected.