Acrylamide Defects the Expression Pattern of the Circadian Clock and

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Food Safety and Toxicology

Acrylamide defects the expression pattern of circadian clock and mitochondrial dynamics in C57BL/6J mice liver and HepG2 cells Xintong Tan, Tong Zhao, Yijie Wang, Jia Wang, Zihan Wang, Zhigang Liu, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02473 • Publication Date (Web): 09 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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Journal of Agricultural and Food Chemistry

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Dear Editor Journal of Agricultural and Food Chemistry May 5, 2018

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Please find attached the manuscript entitled Acrylamide defects the expression pattern of

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circadian clock and mitochondrial dynamics in C57BL/6J mice liver and HepG2 cells by Xintong, Tan;

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Tong, Zhao; Zihan Wang; Jia, Wang; Yijie, Wang; Zhigang, Liu, and myself that we would like to be

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considered for publication in Journal of Agricultural and Food Chemistry.

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Circadian rhythm is a type of oscillator with a cycle of approximately 24 hours and controls a

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variety of physiological and metabolic processes. Accumulated studies during recent years indicated

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that nutrients and some function components played an important role in intervening liver circadian

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clock. But there were few studies to investigate some toxic and harmful substances in food such as

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acrylamide whether disturb the hepatic clocks. In this work, we found that acrylamide could induce

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circadian genes oscillation disorder and down-regulated circadian related protein in mice liver and

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HepG2 cells. Simultaneously, the balances of the daily oscillation of the antioxidant enzymes were

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broken under acrylamide treatment. Furthermore, Acrylamide treatment elevated the mitochondrial

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dynamics genes expressions and influenced the mitochondrial morphology at night phase. It is our hope

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to spur a new discovery of acrylamide toxicity via circadian clock-related mechanism. I hope this paper

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is suitable for Journal of Agricultural and Food Chemistry.

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We deeply appreciate your consideration of our manuscript, and we look forward to receiving

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comments from the reviewers. If you have any queries, please don’t hesitate to contact me at the

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address below. Sincerely yours,

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Xuebo Liu

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PROFESSOR

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ACS Paragon Plus Environment


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COLLEGE OF FOOD SCIENCE AND ENGINEERING,

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NORTHWEST A&F UNIVERSITY, YANGLING, CHINA

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TEL: +86-029-87092325; FAX: +86-029-87092325;

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E-MAIL: [email protected]

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Acrylamide defects the expression pattern of circadian clock and mitochondrial

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dynamics in C57BL/6J mice liver and HepG2 cells

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Xintong, Tan; Tong, Zhao; Zihan Wang; Jia, Wang; Yijie, Wang; Zhigang, Liu and Xuebo, Liu*

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Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and Engineering,

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Northwest A&F University, Yangling, China

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* Corresponding authors: Prof. Xuebo Liu, College of Food Science and Engineering, Northwest A&F University, Xinong Road 2,

Yangling

712100,

China.

Tel:

+862987092817;

Fax:

+86

2987092817;

E-mail:

[email protected] 1

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Abbreviations: ACR, acrylamide; SCN, suprachiasmatic nucleus; Clock, circadian locomotor output cycles kaput; Bmal1, brain and muscle arnt-like protein-1; Cry, cryptochromes; REV-ERB, ;RORs, RAR-related orphan receptors; Per, period; Sirt1, sirtuin 1; HDAC3, Histone deacetylase 3; Dbp, D site of albumin promoter binding protein; ZT, zeitgeber time; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; γ-GT, γ-Glutamyl transferase; ALB, Albumin; POR, P450 oxidoreductase; CYP2E1, Cytochrome P450 2E1; γ-GCS, γ-glutamylcysteine synthetase; GSTA1, glutathione S-transferase A1; GSTP1, glutathione S-transferase P1; Fis1, fission 1; Drp1, dynamin-1-like protein; Mfn1, mitofusin 1; Opa1, optic atrophy 1; Bnip3, BCL2/adenovirus E1B 19 kDa interacting protein 3; Pink1, PTEN induced putative kinase 1; Cox6c, cytochrome c oxidase subunit VIc; Ndufaf4, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex assembly factor 4; NAC, N-acetyl-L-cysteine; 3



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Abstract

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Circadian rhythm helps organisms adapt to their environment and control a variety of

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physiological and metabolic processes. Acrylamide is a toxic compound which can be produced during

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food processing. The aim of this research is to investigate whether circadian clock is involved in the

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toxicity mechanisms of acrylamide in mice liver. Our results revealed that acrylamide markedly induce

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circadian genes oscillation disorder and blocked circadian-related protein in mice liver and HepG2 cells.

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Simultaneously, the balance of the daily oscillation of the antioxidant enzymes was impeded under

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acrylamide treatment. Furthermore, Acrylamide treatment elevated the mitochondrial dynamics genes

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expressions and influenced the mitochondrial morphology at night phase. Acrylamide blocked

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circadian proteins expression via repressing the phosphorylation of AKT or inducing oxidative stress.

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Taken together, our work reveals ACR as a clock-repressing compound generated through Maillard

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browning reaction in certain foods that may possess toxic effect via circadian clock mechanisms.

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Keywords: Acrylamide; Liver; Circadian Clock; Mitochondrial Dynamic; Redox Status;

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Introduction

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Circadian rhythm is a type of oscillator with a cycle of approximately 24 hours. Acquired by

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natural selection during the evolution process, circadian rhythm sets and mediates the physiological

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functions of the organism, maintains synchrony with the environmental cycles of light and nutrients.1, 2

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At organisms level, circadian rhythm is generated by the suprachiasmatic nucleus (SCN), which is the

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major oscillator of the circadian system and organizes clocks in peripheral tissues such as liver, kidney

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and intestine by controlling neural and endocrine pathways. 3, 4 The core mechanism that gives cells the

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ability of circadian oscillations is a cell-autonomous transcriptional-translational feedback loop

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(TTFL).4 In the case of mice, the transcription factors brain and muscle arnt-like protein-1 (Bmal1) and

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circadian locomotor output cycles kaput (Clock) form heterodimers to combine with E-boxes to drive

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the repressors—Cryptochrome (Cry1-2) and Period (Per1-3) transcription. In contrast, the nuclear

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hormone receptors (Rev-erbα and Rev-erβ) and RAR-related orphan receptors (Rorα, Rorβ, and Rorγ)

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regulate the expression of Bmal1 to maintain the rhythm stability and robustness.5, 6 Circadian clock

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controls a number of vital cellular functions, including redox state,

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control.11

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metabolism, and proliferation.9,

Besides cellular functions, approximately 10-25% of the transcriptome is also under circadian

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In mammals, liver is one of critical important organs in various metabolic functions, including the

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processing and uptaking of nutrient, metabolism, and detoxification.12 The transcriptome analysis

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reveals that a series of principal functions of the liver are regulated by circadian clocks. In addition,

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there are many rate-limiting enzymes secreted by liver for nutrient metabolism are expressed in a

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circadian manner.13, 14 Dysfunction of hepatic circadian rhythm, knockout or mutation of some clock

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genes accelerates the progression of multiple liver diseases.15,

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Bmall-knockout had elevated the blood, liver and muscle triglyceride levels and free fatty acids.17

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Compared with wild-type mice, Bmal1+/- and Per2-/- mice had an increased probability of bearing

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spontaneous and radiation-induced liver cancer.18, 19

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For instance, mice with

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Accumulated studies during recent years indicated that nutrients and some function components

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played an important role in intervening liver circadian clock. Mounting evidences showed that

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essential nutrients could regulate peripheral circadian clock genes, including glucose, protein, and

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vitamins.20 Our previous study revealed that some polyphenols like EGCG could improve diet-induced

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metabolic disorder via reprogramming the circadian clock.21 In contrary, numerous food components 5



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could disrupt circadian clock. Research indicated that chronic ethanol consumption could disrupt the

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core molecular clock genes in the liver.22

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Acrylamide (ACR), a water-soluble, toxic, carcinogenic vinyl monomer, has many applications in

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chemical industry and laboratory.23, 24 ACR is one of the Maillard reaction production during food

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processing under high temperature, especially during the process of food which contain asparagine and

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glucose.25, 26 The exposure dose of acrylamide in chemical plant (9500 pmol/g globulin), laboratory

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staff contact gel electrophoresis (54 pmmol/g), and smokers (116 pmol/g) is far higher than the normal

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population of nonsmokers.27, 28 Long-term exposure to even small dosage of acrylamide exhibits toxic

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effect, which may pose a threat to human health.29 In addition, ACR induced oxidative stress and

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apoptosis via interacting with intracellular thiols and glutathione (GSH) cysteine Michael-type adducts.

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Moreover, human daily intake some ACR has potential carcinogenicity risk.26 Noteworthy, these

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adverse effects were implicated with circadian clock. Accordingly, we hypothesis ACR may exert

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hepatotoxicity via defecting liver circadian clock function.

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Above all, the objective of the current research is to uncover the potential influence of ACR on

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the expression of circadian related genes and proteins both in liver and HepG2 cells. Furthermore,

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circadian oscillation of transaminase and anti-oxidase were tested in serum at 6 time points during 24 h

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period. Moreover, we also tested the effects of ACR on mitochondrial morphology and mitochondrial

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dynamics genes expressions at night and light phase, respectively. It is our hope that the present work

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will spur a new discovery of ACR toxicity via circadian clock-related mechanism.

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Materials and methods

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Animals and Diet

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8 weeks-old C57BL/6J mice were bought from Xi’an Jiaotong University (Xi’an, Shaanxi, China).

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ACR (>99%) was purchased from Sigma Aldrich, St Louis, MO, USA. Mice were housed under

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standard conditions (12/12 light-dark cycle, humidity 50 ± 15%, temperature 22 ± 2°C). After 2 weeks’

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adaptive feeding, the experimental animals were randomly divided into two groups (n=40): the control

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group and the ACR group. Control group was fed with a standard diet (AIN-93M, purchased from

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TROPHIC Animal Feed High-tech Co., Ltd. Nantong, China) and pure water, and the mice in ACR

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group were fed with a standard diet and 0.003% ACR in drinking water (w/v) for 16 weeks. Body

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weight, food intake and water intake were recorded weekly. All operations in this experiment strictly

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follow the "Standards for the Protection and Use of Laboratory Animals": the eighth edition, 6


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ISBN-10:0-309-15394-4 and the animal agreement are provided by the animal ethics committee of

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Xi'an Jiaotong University. All surgeries were performed under anesthesia and all efforts were made to

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minimize suffering.

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Analyses of Serum

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The serum total Alanine aminotransferase (ALT) (C009-2), Aspartate aminotransferase (AST)

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(C010-2), Superoxide dismutase (SOD) (A001-3), Albumin (ALB) (A028-1), γ-Glutamyl transferase

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(γ-GT) (C017-2), Reduced glutathione (GSH) (A006-2), Catalase (CAT) (A007-1-1) and

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Malondialdehyde (MDA) (A003-1) levels were measured using enzymatic assay kits (Nanjing

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Jiancheng Bioengineering Institute, Nanjing,China).

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H&E, Immunohistochemical Staining and Electron Microscope Observation

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Histological liver sections, fixed in 4% (v/v) paraformaldehyde/PBS and embedded in paraffin,

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were stained with H&E. The inmmunohistochemichal (IHC) staining was performed as previously

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described.21 Briefly, the fixed liver sections were exposed to the following primary antibodies: Clock

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(ab93804) (1:400; Abcam, Inc, Cambridge, MA, USA), Bmal1 (ab93806) (1:1000; Abcam, Inc., MA,

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USA) and a mouse polyclonal antibody against Sirt1 (23411) (1:200, Cell Signaling Technology, MA,

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USA) at 4 °C overnight. After washing, liver sections were incubated for 20 min at 37 °C with the

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biotinylated goat anti-rabbit or goat anti-mouse diluted in secondary antibody dilution buffer.

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Inmmunohistochemichal (IHC) staining images were acquired using an inverted fluorescent

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microscope (Olympus, Tokyo, Japan) (× 200 ×400) (n = 6/group). For electron microscope observation,

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the liver samples were cut into small pieces (about 1 mm3) and immediately placed in cold PBS (0.1

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mol/L, pH 7.2) containing 2.5% glutaraldehyde at 4 °C until use. Further treatment was performed as

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our previously study.30

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RNA Preparation and qRT-PCR

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The liver tissue and cells mRNA was extracted according to the instructions of Takara mRNA

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extraction kit (TaKaRa MiniBEST Universal RNA Extraction Kit, Dalian, China). The concentration of

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mRNA samples was determined by the Quawell 5000 UV-Vis Spectrophotometer (Quawell Technology,

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San Jose, CA, USA). RNA was stored at -80 °C until use. The RNA (1mg) was inverted to cDNA

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according to the reverse transcription kit (TaKaRa Prime Script RT Master Mix, Dalian, China).

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CFX96TM real-time system (Bio-Rad, Hercules, CA) was used to quantified the mRNA expression.

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Gene-specific mouse and human primers were used as mentioned in Table 1. Ct values were 7



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normalized to GAPDH and β-actin, respectively. The relative gene expression was calculated with the

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2-∆∆Ct method.

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Cell Culture and Treatment

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HepG2 cell line was purchased from Fourth Military Medical University (Xi’an, Shaanxi, China),

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and the medium was RPMI-1640 medium containing 10% fetal bovine serum and 1% double

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antibodies. The cells were cultured in a cell incubator at 5% CO2 and 37 °C.

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Synchronization of Cultured Hepatocytes

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Confluent cells were shocked by 50% horse serum for 2 h and then treated with 5 mM ACR for 24

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h. Then cells were collected for Western blots and qRT-PCR analysis every 4 hours between 24 h and

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44 h time points.

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Cell Viability Assay

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The HepG2 cells were seeded in a 96-well plate (2×104 cells/mL) and incubated in 5% CO2 with

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37 °C. After overnight, the medium was discarded and replaced with serum-free medium containing (or

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without) different concentrations of ACR (0, 0.5, 1, 2.5, 5 and 10.0 mM) for 4 h or 24 h. Then, the

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medium was added to 0.5 mg/mL MTT and incubated for 4 h. After discarding the culture medium, of

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100 mL DMSO was added into each well to dissolve the blue-purple crystals. Finally, the absorbance at

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490 nm wavelengths was measured by use of a microplate reader (Bio-Rad Laboratories, Ltd., China).

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Mitochondrial Membrane Potential (MMP) Assay and Reactive Oxygen Species (ROS)

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Detection

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The HepG2 cells were seeded in a 96-well plate (2×104 cells/mL) and incubated in 5% CO2 with

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37 °C. After overnight, the medium was discarded and replaced with serum-free medium containing (or

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without) different concentrations of ACR (0, 0.5, 1, 2.5, 5 and 10.0 mM) for 4 h or 24 h. For MMP

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assay, after treatment, the culture medium was discarded, and the JC-1 fluorescence staining

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solution with a concentration of l0 µg/mL was added into medium and incubated for 1 h. The

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fluorescence intensity of cells was analyzed under inverted fluorescence microscope (Olympus

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IX71, Tokyo, Japan) (×400). DCFH-DA assay kit (Beyotime, China) was used for ROS detection, after

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treatment, the medium was removed, and the DCFH-DA solution with a total concentration of 10 µM

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was added into 96-well plate and incubated for 0.5 h in the darkness. After washing with PBS, the ROS

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level was observed by inverted fluorescence microscope (Olympus IX71, Tokyo, Japan) (×400).

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Western Blots 8


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The proteins sample of liver tissues from each mice and HepG2 cells were extracted by using

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tissue protein extraction reagent (Thermo Scientific, Prod# 78510) and Cell lysis buffer for western

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(Beyotime, P0013), respectively. SDS-PAGE analysis and immunoblotting measurements were

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performed according to the methods described previously. 31

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Data Analysis

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Data were reported as means ± SEM from at least three independent experiments. Significants

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difference between control and ACR group at six time points were determined by One- way ANOVA

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by Graphpad 6.0 software. Significant differences between mean values with diet (control/ACR in

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drinking water) and two different time points (ZT4 and ZT16) were calculated by Two-way ANOVA,

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followed by Tukey’s test (Graphpad Prism 6.0). Means were considered to be statistically distinct if p

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Acrylamide Defects the Expression Pattern of the Circadian Clock and

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