MeIQx alters autophagosome maturation, cellular lipidomic profiles

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Food Safety and Toxicology

MeIQx alters autophagosome maturation, cellular lipidomic profiles and expression of core pluripotent factors Dan Song, Renpeng Guo, Haibo Huang, Peixiang Zheng, Hong Huang, Xiaoyue Xing, Binran Wang, Jingtong Rong, and Rong Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01041 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Journal of Agricultural and Food Chemistry

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MeIQx alters autophagosome maturation, cellular lipidomic

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profiles and expression of core pluripotent factors

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Dan Song†∇, Renpeng Guo†∇, Haibo Huang†, Peixiang Zheng⊥, Hong Huang†, Qinqin Oyang†, Xiaoyue Xiao⊥, Binran Wang⊥, Jingtong Rong#, Rong Liu*†‡§∥

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∥Department of Internal Medicine, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, United

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⊥Department of Pathogen Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China

†Department of Food Science, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China

‡ National center for international research on animal gut nutrition, Nanjing, China § Jiangsu collaborative innovation center of meat production and processing, Nanjing, China States # Department of Mental Health, Jining Medical University, Jining, China ∇ These authors contributed equally to this work. *Fax: 8625-84396373 E-mail: [email protected]

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Abstract

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MeIQx (2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline), one of the most abundant heterocyclic

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aromatic amines (HAAs) found in human diet, is primarily produced during high temperature meat or

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fish cooking. While MeIQx has been investigated as a potential carcinogen, the cytotoxicity and

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related molecular mechanisms remain unclear. Here we demonstrate that autophagosome maturation

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is blocked by MeIQx. Mechanistically, MeIQx inhibits acidification of lysosomes rather than prevents

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autophagosome-lysosome fusion. Moreover, cellular lipid profiles are altered by MeIQx treatment.

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Notably, many phospholipids and sphingolipids are significantly up-regulated after exposure to

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MeIQx. Furthermore, MeIQx decreases expression of pluripotency-associated proteins in mouse

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embryonic stem cells (ESCs). Together, MeIQx blocks autophagosome maturation through inhibiting

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acidification of lysosomes, alters lipid metabolism and decreases expression of pluripotent factors.

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Our studies provide more cytotoxic evidence and elucidate related mechanisms on the risk of HAAs

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exposure and are expected to promote supervision of food safety and human health.

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Key words: MeIQx, autophagy, lipid metabolism, mouse ESCs

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Introduction

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Heterocyclic aromatic amines (HAAs) are hazardous by-products generated from protein-rich food

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when cooked at high temperature (1-3). HAAs are formed through Maillard reactions among free

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amino acids, reducing sugars and creatine (4). The quantity and type of resulting HAAs depend on

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several factors, such as heating method, temperature, time and the meat material (5). Since first

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discovered in cooked fish by Japanese scientists in 1997, now more than 30 different kinds of HAAs

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have been isolated and identified (6). These compounds have been extensively investigated as

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mutagens and carcinogens (7, 8).

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MeIQx (2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline) is one of the most abundant HAAs

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found in human diet (9). In mice and rats models, MeIQx could induce mainly liver tumors (10, 11).

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In clinical studies, exposure to MeIQx is associated with an increment of colorectal cancer risk (12).

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Also, MeIQx is thought to be causative agents for human pancreatic cancer in some studies (13, 14).

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When ingested by cells, MeIQx and other HAAs can be bio-activated through N-oxidation of the

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exocyclic amine group to produce N-hydroxyderivatives, which are mutagenic (15). These derivatives

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and some intermediates lead to DNA strand breaks, chromosomal aberrations, mutations and final

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carcinogenicity (16). Further, MeIQx is reported to induce oxidative stress and apoptosis in human

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hepatoma cells (17). Overall, the genotoxicity and cytotoxicity caused by MeIQx and other HAAs

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have attracted increasing attentions.

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Autophagy is a highly conserved catabolic process in eukaryote cells. It is essential for cellular

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homeostasis through elimination and recycling of large cytoplasmic components, such as abnormal

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protein aggregates and damaged organelles, via lysosomal degradation (18). Autophagy is a highly

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dynamic process involving initiation, nucleation and extension, maturation of the autophagosomes,

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degradation, and reformation of autophagolysosomes (19). In this process, the isolation membrane of

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phagophore first recruits multiple proteins and expands to form a double-membrane autophagosome,

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which will then fuse with lysosomes to generate autolysosomes where sequestrated materials are

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degraded (20). Autophagy plays crucial roles in the progress of both physiological and pathological ACS Paragon Plus Environment


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conditions, such as tumorigenesis, neurodegenerative diseases and metabolic disorders (21). Whether

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harmful substances produced during food processing will affect cellular autophagy remains to be

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investigated.

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Embryonic stem cells (ESCs) are pluripotent, which means that ESCs can self-renew indefinitely

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under optimal culture conditions and have the ability of forming all cell types (22). Thus, ESCs are

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promising donor cell sources for regenerative medicine. The pluripotency of ESCs is mainly

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maintained by a feed-forward networks formed by transcription factors Oct4, Sox2, and Nanog (23,

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24). Quantitative balances of these factors are necessary for unlimited self-renewal of ESCs.

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Increasing expression of Oct4 leads to differentiation of mouse ESCs into primitive endoderm and

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mesoderm (25, 26). Overexpression of Nanog results in Leukemia Inhibitory Factor-independent self-

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renewal in mouse ESCs, while deficiency of Nanog induces differentiation of ESCs into

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extraembryonic endoderm lineage (27).

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In this study, we investigate the effects of MeIQx on autophagy and find that MeIQx can noticeably

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block autophagy flux by inhibiting maturation of autolysosomes. Also, lipidomic profiles of cultured

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hepatocytes are disturbed by MeIQx with up-regulated levels of phospholipids and sphingolipids in

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comparison with control groups. Further, our study indicates that MeIQx treatment represses

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expression of core transcription factors in mouse ESCs, which may impair pluripotency of ESCs

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during long-term culture.

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Materials and Methods

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Reagents and antibodies

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MeIQx (≥99%) was obtained from Toronto Research Chemicals Inc. (Toronto, Canada), autophagy

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inducer Torin2 (SML124), autophagy inhibitor Chloroquine (CQ, C6628) and methanol were

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purchased from Sigma Aldrich Co. (St. Louis, USA). Chloroform and ethanol were procured from the

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office of laboratory and equipment management of Nanjing Agricultural University. All solvents for

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hydrophilic and lipid extraction were of chromatographic grade. Others were of analytical grade.


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Antibodies against β-tubulin (T8328) and LC3 (L7543) were purchased from Sigma Aldrich Co. (St.

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Louis, USA), and p62 (ab109012) was purchased from Abcam (Cambridge, UK). IgG-HRP

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conjugated anti-rabbit or mouse secondary antibodies were purchased from Sangon Biotech (Shanghai,

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China). Secondary antibodies conjugated to Alexa Fluor dyes were purchased from Jackson

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ImmunoResearch (West Grove, USA).

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Cell culture and transfection

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The human hepatocytes HL-7702 were cultured in a RPMI 1640 from Corning Cellgro® (Manassas,

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USA) medium containing 10% fetal bovine serum (FBS, PAN, Adenbach, Germany) and U2OS cells

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were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, USA)

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supplemented with 10% FBS, 1% Penicillin-Streptomycin solution (Hyclone, Logan, USA). J1 ESCs

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were cultured on gelatin-treated plates in ES cell culture medium consisting of knockout DMEM

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supplemented with 15% FBS (ES quality, Hyclone, Logan, USA), 1000 U/ml leukemia inhibitory

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factor (LIF) (ESGRO, Chemicon International Inc., Temecula,

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acids (Sigma Aldrich Co., St. Louis, USA), 0.1 mM β-mercaptoethanol (Sigma Aldrich Co., St. Louis,

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USA), 1 mM L-glutamine (Thermo Fisher Scientific Inc., Waltham, USA), and penicillin (100 U/ml)

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and streptomycin (100 μg/ml) (Thermo Fisher Scientific Inc., Waltham, USA). All cultured cells were

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maintained at 37 °C with 5% CO2 under a humidified atmosphere. The majority of experiments were

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performed using U2OS cells, and human hepatocytes were used for metabolomic detection, and J1

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ESCs were cultured to test the effects of MeIQx exposure on expression of pluripotent factors. For

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MeIQx treatment, MeIQx was dissolved in sterilized deionized water to obtain a stock solution of 1

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M and stored at -20℃，and then diluted to the desired concentration.

USA), 0.1 mM non-essential amino

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mRFP-GFP-LC3 assay

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Autophagy flux was assessed through mRFP-GFP-LC3 adenovirus probe assay. U2OS cells were

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cultured and transfected with the mRFP-GFP-LC3 adenovirus. Twenty-four hours after transfection,



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the cells were treated with MeIQX (1 µM) for another 6 h. Then the cells were fixed with 4%

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paraformaldehyde and images were obtained using a laser scanning confocal microscope (Olympus

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FV3000, Tokyo, Japan) under 60× magnification. The yellow spots indicated autophagosomes and the

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red spots indicated autolysosomes.

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Immunofluorescence staining

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U2OS cells grown on coverslips were fixed in 4% paraformaldehyde at room temperature for 10

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min after rinsing with phosphate buffered saline (PBS) three times. Then the cells were washed three

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times with sterilized PBS and incubated with primary antibodies diluted in blocking buffer (1% bovine

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serum albumin (BSA), 0.1% Triton X-100 in PBS) for overnight at 4 °C in a humidified chamber.

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Cells were washed with PBS three times with 10 min each, and then incubated with Alexa Fluor-

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conjugated secondary antibody (Invitrogen, Carlsbad, USA) at room temperature for 1 hour.

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Coverslips were mounted and fluorescence images were obtained using a laser scanning confocal

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microscope (Olympus FV3000, Tokyo, Japan).

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LysoTracker staining

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LysoTracker staining were performed as previously described (28). U2OS cells were seeded on

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Lab-TekChambered cover-glass bottom dish and treated with 1 µM MeIQX for 6 h, and then incubated

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with 1 µM LysoTracker Red DND-99 for the last 30 minutes. Lysosomes indicated by LysoTracker

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staining were observed by a laser scanning confocal microscope (Olympus FV3000, Tokyo, Japan).

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Western blotting

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The treated U2OS or J1 ESC protein samples were collected and denatured with sodium dodecyl

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sulfate (SDS) loading buffer and boiled at 100 °C for 10 min. Then samples were separated by SDS-

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PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Biorad, Hercules, USA). The

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membrane was incubated with primary antibodies with 5% BSA overnight at 4℃. Then the membrane


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was incubated with HRP-conjugated secondary antibodies at 1: 5000 dilutions for 30 min at room

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temperature. The signal was detected using an enhanced chemiluminescence detection regents and

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densitometric analysis of bands was quantified with Image J software.

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RNA extraction and Quantitative real-time PCR

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Total RNA was extracted from ESCs using RNAiso plus and cDNA was synthesized using a

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PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Kusatsu, Japan). Quantitative real-time

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PCR was conducted on a CFX 96TM real-time system instrument with an iTaq Univer SYBR Green

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Supermix (Bio-Rad, Hercules, USA). GAPDH was used as an internal control. Primer sequences were

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shown in Supplementary Table 1. Each reaction was performed in triplicate.

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Lipid metabolite extraction from cells

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The lipid metabolite extraction was performed according to the Folch et al. (29) described method

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with minor modification. The 4 ml of extraction buffer (chloroform/methanol at 2:1, v/v) was added

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to cells suspended in 1 ml of Dulbecco's Phosphate Buffered Saline (DPBS, Hyclone, Logan, USA)

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buffer in glass tubes. The mixture was vortexed and stirred for a few minutes and then centrifuged at

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600 g for 15 min at room temperature. Subsequently, the lower organic phase was collected using

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glass syringe to a fresh glass tube and dried by nitrogen, then the dried samples were kept in dry ice

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for detection.

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UPLC-Q-Exactive Orbitrap/MS methods for lipid

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Lipid analyses were conducted on the UPLC-Q-Exactive Orbitrap mass spectrometer (Thermo

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Fisher Scientific Inc., Waltham, USA) equipped with a heated electrospray ionozation (HESI) probe.

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Lipid extracts were separated by a Cortecs C18 100 × 2.1 mm column (Waters). A binary solvent

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system including mobile phase A (ACN: H2O (60:40), 10 mM ammonium acetate) and mobile phase

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B (IPA: ACN (90:10), 10 mM ammonium acetate) was used to elute sample with a flow rate of 220



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µL/min as follows: 37% B to 98% B, 20 min; 98% B, 8 min; re-equilibration with 37% B, 7 min.

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Colum chamber and sample tray were held at 40 °C and 10 °C, respectively. The data with mass range

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of m/z 240-2000 and 200-2000 were obtained through dependent MS acquisition in both negative and

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positive ion mode, respectively. The full scan and fragment spectra were obtained with resolution of

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70,000 and 17,500, respectively. The detailed parameters were set as below: spray voltage: 3000v;

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capillary temperature: 320 °C; heater temperature: 300 °C; sheath gas flow rate: 35 Arb; auxiliary gas

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flow rate: 10 Arb. The software Lipid search (Thermo Fisher Scientific Inc., Waltham, USA) was used

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for data analysis and lipid identification. For lipid analysis, only chromatographic area >5E6 was

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regarded as a reliable lipid identification methods and lipids were identified based on MS2, with a

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MS1 mass error of

MeIQx alters autophagosome maturation, cellular lipidomic profiles

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