Transcriptional and proteomic analysis revealed a synergistic effect of

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Transcriptional and proteomic analysis revealed a synergistic effect of aflatoxin M1 and ochratoxin A mycotoxins on intestinal epithelial integrity of differentiated human Caco-2 cells Yanan Gao, Songli Li, Xiaoyu Bao, Chaochao Luo, Huaigu Yang, Jiaqi Wang, Shengguo Zhao, and Nan Zheng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00241 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018

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Journal of Proteome Research

Transcriptional and proteomic analysis revealed a synergistic effect of aflatoxin M1 and ochratoxin A mycotoxins on intestinal epithelial integrity of differentiated human Caco-2 cells † † Yanan Gao1,2,3,4, , Songli Li1,2,3,4, , Xiaoyu Bao1,2,3,4, Chaochao Luo1,2,3,4, Huaigu

∗

Yang1,2,3,4, Jiaqi Wang1,2,3,4, Shengguo Zhao1,2,3,4, Nan Zheng1,2,3,4, 1

Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry

of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China 2

Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of

Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China 3

Milk and Dairy Product Inspection Center of Ministry of Agriculture, Beijing 100193,

P.R. China 4

State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese

Academy of Agricultural Sciences, Beijing 100193, P. R. China

†

These authors (Yanan Gao, Songli Li) contributed equally.

*Corresponding author: Nan Zheng, Ph.D. Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, P. R. China. Tel: +86-10-62816069; Fax: +86-10-62897587 E-mail: [email protected] 1

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Abstract Aflatoxin M1 (AFM1) is a common mycotoxin in dairy milk and it is typically concurrently present with other mycotoxins that may represent a threat for food safety. However, knowledge on how AFM1, alone or in combination with other mycotoxins may affect human intestinal epithelial integrity remain to be established. We employed transcriptome and proteome analysis integrated with biological validation to reveal the molecular basis underlining the effect AFM1 and/or ochratoxin A (OTA) exposure on intestinal epithelial integrity of differentiated Caco-2 cells. Exposure to 4 µg/ml of OTA was found to disrupt human gut epithelial integrity, whereas 4 µg/ml of AFM1 did not. Integrated transcriptome and proteome analysis of AFM1 and OTA, alone or in combination, indicate synergistic effect of the two mycotoxins in disrupting intestinal integrity. This effect was mechanistically linked to a broad ranges of pathways related to intestinal integrity enriched by down-regulated genes and proteins, associated to focal adhesion, adherens junction, and gap junction pathways. Furthermore, the cross–omics analysis of mixed AFM1 and OTA compared with OTA alone suggest that kinases family members, including MLCK, MAPKs, and PKC are the potential key regulators on modulating intestinal epithelial integrity. These findings provide novel insight into the synergistic detrimental role of multiple mycotoxins in disrupting intestinal integrity, and, therefore, identify potential target to improve milk safety related to human health. Transcriptomic raw data is available on Sequence Read Archive (SRA) database and the SRA accession is SRP133808 (https://www.ncbi.nlm.nih.gov/sra/SRP133808). The mass spectrometry proteomics 2


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data

have

been

deposited

to

the

ProteomeXchange

Consortium

(http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD009437 and 10.6019/PXD009437. Key words: aflatoxin M1, ochratoxin A, omics, intestinal integrity, synergistic effect.

Introduction Mycotoxins are secondary metabolites produced by a wide range of filamentous fungi, and are likely to cause toxic responses in both human and animals.1 Usually, toxins present not only in the air or dust but also in foods, including animal products such as meat, milk and eggs, thereby raising the possibility of daily mycotoxins exposure.2, 3 Cow’s milk and its derivatives account for a high proportion of human diet at all ages as necessary source for macro- and micronutrients to sustain growth and maintain health.4 The milk contamination by mycotoxins however is a tangible potential health issue, especially for children who are more susceptible to toxic compounds. Specifically, studies on aflatoxin M1 (AFM1), one of the most intensively studied mycotoxin, help setting a maximum residue limit (MRL) in milk, however the content of AFM1 in milk is far exceeding the MRL level in EU (0.05 µg/kg).1 The maximum concentration of AFM1 in the raw cow milk collected from Albania is 0.85 µg/kg and it is exceeding the MRL level in China (0.5 µg/kg).5 In Africa, it is common to find high concentrations of AFM1 in milk. The detection of AFM1 in 13 milk samples from Egypt revealed concentrations ranging from 5.0 to 8.0 µg/L.6 In Nigeria, a study performed in 2006 on 22 raw milk samples found that the range of AFM1 levels was 3



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2.04-4.0 µg/L.7 The other mycotoxins including ochratoxin A (OTA), zearalenone (ZEA) and its metabolites, have also been found in milk samples, yet their MRL levels have not been established.8 Aflatoxin consumption could cause some human diseases, or aflatoxicoses, due to the potential roles of toxicity and carcinogenicity.9 Acute aflatoxicosis has high toxicity that can result in death, and chronic aflatoxicosis leads to cancer, immune suppression, and other “slow progressing” pathological conditions.9 Mycotoxin OTA, ZEA and others are reported to increase the susceptibility of animals to infectious disease.2 The frequent and global occurrence of mycotoxins in milk has become a serious food safety issue. More importantly, it’s more likely to find the co-presence of AFM1 and OTA in milk products. In the northwest of French, the raw milk was tested positive to the occurrence of AFM1 and OTA.10 And these two mycotoxins were also detected in the baby foods in Portugal, including flours and milk powder.11 However, to date, only AFM1 has its MRL regulation in milk products.8 The few available study demonstrated that AFM1 and OTA performed similar higher cytotoxicity in human intestinal Caco-2 cells, followed by ZEA and α-ZOL.12 This finding suggests that it’s likely to underestimate the significance of OTA on human public health. It is therefore reasonable to evaluate the toxicity of OTA through considering AFM1 as its benchmark. Most of the mycotoxins adverse effects reported above are mostly related to their effects on the intestinal tract. Intestinal epithelial cells (IECs) composing the largest human barrier exposed to the external environment, are exposed to a high concentration of mycotoxin ingested with contaminated milk.13,14 IECs are principally maintained by 4


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well-organized intercellular structures (junctional complexes) constituted by tight junctions, adherence junctions, desmosomes, gap junctions, and integrin, which surround the apical region of epithelial cells.15 The changes of junctional complexes can be considered as the indicators of the modulation of barrier properties induced by a variety of stimuli, including toxic substances.16 Deoxynivalenol (DON) could increase the paracellular flux of lucifer yellow (LY) and FITC-dextran (4 kDa) and decrease the expression levels of claudin-1, claudin-3, and claudin-4, thus disrupting the intestinal epithelial integrity of human.17 Hence, the evaluation of intestinal integrity is crucial in assessing risk subsequence to food contaminant exposure. The multi-exposure to mycotoxins is the most common scenario in milk.8 The simultaneous presence of mycotoxins in food may be more toxic than the presence of single mycotoxins and may lead to different interactive effects, such as additive, synergistic, or antagonistic effects.18 Hence, it seems more logical to assess the toxicological effects of certain mycotoxins in combination rather than singularly. Recently, some works have applied transcriptome and proteome analysis to evaluate the effect on growth hormone suppression triggered by mycotoxin T-2 in rat-derived GH3 cells or the early hepatotoxicity induced by OTA in specific pathogen-free male F344 rats.19,20 However, the effect of combined mycotoxins still remains to be investigated, with the exception of one work that established the effect of combined DON and ZEA using metabolic profiling in serum and liver of mice.21 However, the mechanistic insight into the interactive effects on the intestinal integrity induced by multi mycotoxins present in milk is still lacking. To fill this knowledge gap, we 5



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compared the transcriptome and proteome of differentiated Caco-2 cells treated with individual AFM1, OTA and their combination (AFM1+OTA). After culturing them for 16-22 days, fully differentiated Caco-2 cells exhibiting the characteristics of mature enterocytes with a dome-like appearance can be obtained.22,23 Moreover, to confirm the interactive effects of AFM1 and OTA on intestinal integrity, in terms of additive, synergistic or antagonist toxicity, the pathways enriched by differentially expressed genes (DEGs) and proteins in AFM1+OTA compared with OTA alone treatment were biologically validated. This study provides new insights into the underlying molecular mechanisms of the disruption of intestinal integrity induced by AFM1 and OTA. Materials and methods Toxins AFM1 (C17H12O7; molecular weight: 328) and OTA (C20H18ClNO6; molecular weight: 403) were purchased from Fermentek (Jerusalem, Israel). Mycotoxins were dissolved in methanol as previously described.24,25 AFM1 and OTA were dissolved in methanol to concentrations of 400 µg/ml and 5000 µg/ml, respectively. Both stock solutions were stored at -20 °C for later use. The final concentrations of AFM1 (0.00005, 0.0005, 0.005, 0.05, 0.25, 0.5, 1, 2 and 4 µg/ml) and OTA (0.00005, 0.0005, 0.005, 0.05, 0.25, 0.5, 1, 2 and 4 µg/ml) were prepared by adding Dulbecco’s modified Eagle medium (DMEM, Life Technologies, Carlsbad, CA, USA). The final methanol concentration in the medium was less than 1% (v/v), which could not affect the normal growth of cells. Methanol at a final concentration of 1% (v/v) was used as the vehicle control in all experiments. A concentration of 0.00005 and 0.0005 µg/ml of 6


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AFM1 used in this study was referred to the MRL in EU (0.05 µg/kg) and China (0.5 µg/kg), respectively. Cell lines and culture The human colon adenocarcinoma Caco-2 cell line (passage number, 18) was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). In the present study, Caco-2 cells were used at a passage number between 23 and 31. Caco-2 cells were cultured in 6- or 12-well transwell chambers (Corning, NY, USA) at a density of 4 × 104 cells/cm2 in DMEM containing 4.5 g/l glucose, 10% fetal bovine serum (Gibco, CA, USA), antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin, Gibco, CA, USA), and 1% nonessential amino acids (NEAA; Gibco, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every other day until 21 days to form a differentiated Caco-2 cells monolayer. 26

In the present study, the mean transepithelial electrical resistance (TEER) values at

baseline was 802 ± 110 Ω·cm2 as measured by a Millicell-ERS volt-ohm meter (Millipore, Temecula, CA, USA). Analysis of cell viability The effects of mycotoxins on the proliferation of intestinal cells were determined using the Enhanced Cell Counting Kit (CCK)-8 (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In brief, AFM1 (0.00005, 0.0005, 0.005, 0.05, 0.25, 0.5, 1, 2 and 4 µg/ml) and OTA (0.00005, 0.0005, 0.005, 0.05, 0.25, 0.5, 1, 2 and 4 µg/ml) were added to apical and basal compartments of transwell chambers, exposure to both apical and basolateral side mimics the in vivo situation. After 48 h, 7



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cells were rinsed with PBS and then 100 µl of CCK-8 solution (10 in 90 µl serum-free medium) was added, and cells were then incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using an automated ELISA reader (Thermo Scientific, Waltham, MA, USA). Three biological replicates were produced. Transepithelial electrical resistance (TEER) measurement Differentiated Caco-2 cells were challenged for 48 h with gradient concentrations of AFM1 and OTA as did in the analysis of cell viability. Additionally, 4 µg/ml AFM1 and 4 µg/ml OTA were chosen to constitute their combination on the basis of the cell viability and TEER values of individual AFM1 and OTA treatment. Individual and combined mycotoxins were also added to apical and basal compartments of transwell chambers. Results were expressed as change (%) relative to the initial TEER value for each insert and presented as the mean ± standard error of the mean (S.E.M.) of five independent experiments. RNA-seq, data processing, and gene annotation RNA of the differentiated Caco-2 cells without mycotoxins (Control) and cells treated with individual AFM1, OTA and the combination of AFM1 and OTA were extracted using the kits (Tiangen Biotechnology Co., LTD, China) according to the manufacturer’s protocol. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads, while prokaryotic mRNA was enriched by removing rRNA using Ribo-ZeroTM Magnetic Kit (Epicentre). The enriched mRNA was then fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA 8


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polymerase I, RNase H, dNTP and buffer. Afterwards, the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly (A) added, and ligated to Illumina sequencing adapters. The ligation products were selected based on size by agarose gel electrophoresis, PCR amplication, and sequencing were done using Illumina HiSeq 2500 (Illumina, San Diego, USA). High quality reads (clean reads) were obtained by removing low-quality reads and ribosome RNA mapped reads, followed by mapping to the reference genome of human usingTopHat2.27 The gene expression level was normalized by using FPKM (Fragments Per Kilobase of transcript per Million mapped reads). The genes with a fold change ≥2 and a false discovery rate (FDR) 1.2 or < 0.83 were considered to be differentially regulated on the basis of previous reports.29, 30

The

mass

spectrometry

proteomics

data

have

been

deposited

to

the

ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE

partner

repository

with

the

dataset

identifier

PXD009437

and 13



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10.6019/PXD009437. Western blotting assays To verify the differentially expressed proteins, western blotting assays were conducted. Caco-2 cell monolayers were cultured on transwell chambers and incubated for 48 h with the addition of OTA or the combination of AFM1 and OTA, which were added to apical and basal compartments. Caco-2 cells were lysed with RIPA lysis buffer (Beyotime

Biotechnology,

Beijing,

China)

containing

protease

inhibitors

(Sigma-Aldrich). Equal amounts of protein were loaded on sodium dodecyl sulfate (SDS)-polyacrylamide

gels,

and

transferred

onto

polyvinylidene

difluoride

membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk in PBS for 1.5 h at room temperature. Subsequently, rabbit anti-connexin 43 (Abcam, Cambridge, MA, USA), mouse anti-PKC (Abcam), rabbit anti-FABP6 antibody (Abcam), rabbit anti-Bax inhibitor 1 (Abcam), and rabbit anti-β-actin (Cell Signaling Technology, MA, USA) antibodies were diluted according to the manufacturers’ instructions and incubated with membranes for 3 h at room temperature (RT). Goat anti-rabbit and goat anti-mouse IgG conjugated to horseradish peroxidase (Bioss Antibodies, Beijing, China) was applied for 1 h at RT. Peroxidase activity was visualized on a radiographic film using enhanced chemiluminescence reagents (Thermo Scientific, Waltham, MA, USA). Signal intensities were determined by densitometry using ImageJ 2× software (Version 2.1.0, National Institutes of Health, Bethesda, MD, USA, 2006), and values were normalized to the loading control (β-actin). The results of western blotting are expressed as mean ± SEM of 14


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three independent experiments. Protein-Protein Interaction (PPI) Network analysis PPI networks were generated from the DEGs and differentially expressed proteins associated with intestinal integrity. The PPI interactions with a combined score (0: lowest confidence; 1: highest confidence) larger than 0.7 were used for further network analysis. All differentially expressed proteins were mapped onto the PPI network and visualized by Cytoscape. The BiNGO plugin, a Cytoscape plugin, was used to retrieve the GO terms. Validation of down-regulate activity of PKC in intestinal integrity by small interfering RNA (siRNA) Based on the transcriptome and proteome analysis, protein kinase C (PKC) was supposed to modulate intestinal integrity in differentiated Caco-2 cells induced by the combination of AFM1 and OTA. To confirm this, the expression level of PKC-α gene was down-regulated by siRNA. Hence, PKC-αsiRNA (PKCsiRNA; GenePharma) and negative control (NC) siRNA were transfected into differentiated Caco-2 cells using OptiMEM siRNA transfection medium and Lipofectin 2000 siRNA transfection reagent according to the manufacturer’s instructions. The primer sequences were GCACAGGAUCAGCUAUCAATT (sense) and UUGAUAGCUGAUCCUGUGCTT (antisense). The transfection medium containing the siRNA and siRNA transfection reagent was mixed with serum-free culture medium and incubated with the differentiated Caco-2 cells. The medium was replaced after 6 h, and cells were incubated for an additional 24 h, and then TEER values were determined as described 15



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above. Cells were then collected and lysed, and PKC-α and connexin protein expression was examined. Results Effect of AFM1 and OTA on the viability and TEER values of Caco-2 cell monolayers To evaluate the cytotoxicity of AFM1 and OTA on differentiated Caco-2 cells, cell viability was monitored. No decreased proliferation with increasing mycotoxins concentrations was observed for both mycotoxins tested. Both AFM1 and OTA were not cytotoxic to differentiated Caco-2 cells within the concentration ranging between 0.00005-4 µg/ml when cells were treated with one single mycotoxin (Figure 1A). Furthermore, when investigating the effect of AFM1 and OTA on intestinal epithelial barrier function, AFM1 had no negative effect on TEER values, whereas OTA treated cells clearly decreased intestinal integrity in a dose-dependent manner (Figure 1B). A 4 µg/ml of OTA was found a potential to disrupt epithelial integrity, whereas 4 µg/ml of AFM1 were not. The TEER values of Caco-2 cells treated with combined AFM1 and OTA at 4 µg/ml were significantly lower than the TEER values when those cells were treated with OTA alone at 4 µg/ml, suggesting a potential synergistic effect on intestinal integrity by the combination of AFM1 and OTA (Figure 1C). Transcriptome analysis of differentiated Caco-2 cells induced by AFM1 and OTA Total number of transcripts detected in control, AFM1, OTA, and AFM1+OTA group was 19199, 18873, 20416, and 19797, respectively. To define the gene variation between mycotoxins-treated and control groups, the transcriptome was compared 16


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between individual or combined mycotoxins and control groups. When Caco-2 cells were treated with AFM1 alone at 4 µg/ml, 23 up-regulated and 476 down-regulated genes compared with control group were identified. When cells treated with 4 µg/ml of OTA were compared with untreated cells, 1437 up-regulated and 7965 down-regulated proteins were identified. Furthermore, when AFM1+OTA group were compared to control, there were 902 up-regulated and 10,004 down-regulated genes. Some intestinal integrity-related pathways, namely gap junction, focal adhesion, tight junction, adherens junction, and regulation of actin cytoskeleton were involved in the both individual and combined mycotoxins-treated groups (Table S-2). More importantly, the number of DEGs in the combination treatment was more than OTA treatment, followed by the AFM1 treatment (Table S-2). Therefore, the omics effect of AFM1 and OTA on the intestinal integrity of Caco-2 cells is consistent with our phenotypic results related to gut barrier integrity induced by these mycotoxins (Figure 1C). To further study the underlying molecular details of the synergistic effect on intestinal integrity induced by combined AFM1 and OTA, the transcriptome profile of Caco-2 cells treated by the combination of AFM1 and OTA (AFM1+OTA) was compared with those treated with OTA alone. About 27 and 29 million 150-bp paired-end clean reads were obtained from OTA and AFM1+OTA group, respectively. Using the splice-aware aligner Tophat2, 85.96% and 87.83% of reads were mapped to the human genome in the two treatment groups, respectively. Importantly, both in OTA and in AFM1+OTA groups, more than 95% of reads were mapped to the exon region, 17



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including 5’ untranslated region (UTR) and 3’ UTR region (Figure 2A), suggesting that our RNA-seq data could precisely depict the transcription of protein coding genes in these treatments. To identify genes involved in AFM1+OTA compared to the OTA group, DEGs with a fold change ≥2 and a FDR

Transcriptional and proteomic analysis revealed a synergistic effect of

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