IgY Reduces AFB1-Induced Cytotoxicity, Cellular Dysfunction, and

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IgY reduces AFB1-induced cytotoxicity, cellular dysfunction, and genotoxicity in human L-02 hepatocytes and Swan 71 trophoblasts Taotao Qiu, Xing Shen, Zhen Tian, Riming Huang, Xiangmei Li, Juan Wang, Rong Wang, Yuanming Sun, Yiguo Jiang, Huidong Zhang, and Hongtao Lei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05385 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

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Journal of Agricultural and Food Chemistry

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IgY reduces AFB1-induced cytotoxicity, cellular dysfunction, and

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genotoxicity in human L-02 hepatocytes and Swan 71 trophoblasts

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Taotao Qiu†, §, Xing Shen†, Zhen Tian§, Riming Huang†, Xiangmei Li†, Juan Wang†,

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Rong Wang§, Yuanming Sun†, Yiguo Jiang⊥, Hongtao Lei*, † and Huidong Zhang*, §

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†

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University, Guangzhou, 510642, China.

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§

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University, No.17 People's South Road, Chengdu, 610041, China.

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Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural

Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan

⊥

Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu District,

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Guangzhou, China.

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*

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Phone: +862085283925. Fax: +862085280270

E-mails: [email protected] (H.D. Zhang); [email protected] (H.T. Lei).

ACS Paragon Plus Environment


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Abstract:

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Aflatoxin B1 (AFB1) causes hepatotoxic, genotoxic, and immunotoxic effects in

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a variety of species. Although various neutralizing agents of AFB1 toxicity have been

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studied, the egg yolk immunoglobulins (IgY) detoxification of small molecular toxins

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and the mechanisms underlying such effects have not yet been reported. In this

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investigation, anti-AFB1 IgY against AFB1 was successfully raised, and a competitive

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indirect enzyme-linked immunosorbent assay was established with a sensitive

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half-maximal inhibitory concentration (IC50, 2.4 ng/mL) and dynamic working range

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(0.13–43.0 ng/mL). The anti-AFB1 IgY obtained reduced AFB1-induced cytotoxicity,

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cellular dysfunction, and genotoxicity by protecting cells against apoptotic body

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formation and DNA strand breaks, preventing G2/M phase cell cycle arrest, reducing

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AFB1-DNA adduct and ROS production, and maintaining cell migration and invasion

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and the mitochondrial membrane potential. Anti-AFB1 IgY significantly inhibited the

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AFB1-induced expression of proteins related to antioxidative, pro-apoptotic, and

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anti-apoptotic processes in a strong dose-dependent manner. These experiments

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demonstrated that the anti-AFB1 IgY-bound AFB1 could not enter cells. This is the

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first time that IgY has been found to reduce the effects of small molecular toxins,

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which will be beneficial for the development of antibodies as detoxication agents.

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Keywords: IgY, aflatoxin B1, detoxification, cytotoxicity, cellular dysfunction,

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genotoxicity

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INTRODUCTION Aflatoxins, the secondary metabolites produced by Aspergillus flavus and

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Aspergillus parasiticus, are widely distributed in various food products and

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simultaneously harm multiple human organs by producing a cascade of responses in

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the affected individual.1 Among these aflatoxins, aflatoxin B1 (AFB1) is the most

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hepatotoxic, genotoxic and immunotoxic to humans and animals.2 AFB1 is classified

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as a Group 1 human carcinogen and has no recognized safe dose by the International

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Agency for Research on Cancer.3 In cells, AFB1 is metabolized to AFB1

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exo-8,9-epoxide (exo-AFBO),4 which binds to nuclear DNA and thus forms DNA

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adducts5 that can ultimately lead to mitochondrial swelling, reduce mitochondrial

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activity and ATP content, and induce cellular senescence and catastrophic mutation or

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apoptosis.6 It is possible that cellular dysfunction may be due to AFB1-induced

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alterations of cellular macromolecules.7 The toxic effect of AFB1 may also be

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attributed to its enhancement of the reactive oxygen species (ROS) level in living

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cells, which may attack lipids, proteins, and nucleic acids.8 Although many methods,

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including physical, chemical, and biological treatments, have been used to degrade

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AFB1 to improve food safety,9,10 most of these processes lead to the formation of toxic

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residues or derivatives and a reduction in the food’s nutritional values and

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organoleptic qualities.11

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Egg yolk immunoglobulins (IgY), an antibody generated from chickens, is

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composed of two light chains and two heavy chains, like mammalian counterpart

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IgG.12 However, in contrast, IgY can neither activate the mammalian complement 3



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system nor interact with human Fc receptors and mammalian rheumatoid factors.13

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Additionally, chickens can produce a strong immune response against mammalian

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antigens owing to the phylogenetic distance between mammals and birds.14 Because

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of these advantages, IgY has been used for the treatment of various gastroenteric

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infectious diseases,15 and against macromolecular protein toxins, such as Shiga

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toxin,16 ricin toxin,17 botulinum toxins,18 and viper venom.19 However, the IgY

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detoxification of small molecular toxins and the mechanism behind this effect have

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not yet been reported.

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In this study, AFB1 was conjugated to bovine serum albumin (BSA), in the

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presence of excess glycolic acid (GA) as a spacer, for use as an immunizing antigen

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(AFB1-GA-BSA) to raise IgY antibodies against in chicken. After evaluation of the

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sensitivity and specificity of the IgY obtained, its detoxification ability was

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investigated. This work represents the first time that an IgY has been found to be

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capable of inhibiting AFB1-induced cytotoxicity, cellular dysfunction, and

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genotoxicity in two typical human cell lines: L-02 hepatocytes and Swan 71

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trophoblasts.

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MATERIALS AND METHODS

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Chemicals and Reagents

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Aflatoxin B1 was purchased from J&K Scientific (Beijing, China). Bovine serum

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albumin (BSA), ovalbumin (OVA), Freund’s incomplete adjuvant (FIA), Freund’s

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complete adjuvant (FCA), dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), 4


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1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),

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N-hydroxysuccinimide (NHS), N,N-dimethylformamide (DMF),

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3,3′,5,5′-tetramethylbenzidine (TMB), and horseradish peroxidase (HRP)-conjugated

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goat anti-rabbit IgG secondary antibody were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). Anti- Zearalenone (ZEN) IgY was prepared at Guangdong

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Provincial Key Laboratory of Food Quality and Safety (Guangzhou, China).

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Antibodies against for superoxide dismutase 1 (SOD1), catalase (CAT), apoptosis

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regulator (BCL2), survivin, caspase-3 and BCL2 associated X, apoptosis regulator

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(BAX) were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA).

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All ingredients for the culture medium were purchased from HyClone (Logan, UT,

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USA). The apoptosis detection kit, 2′,7′-dichlorofluorescein diacetate, JC-1 monomers,

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cell cycle analysis kit, and Hoechst 33342 stain were purchased from Beyotime

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(Shanghai, China). The AFB1-DNA ELISA kit was purchased from Sinogeneclon Co.,

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Ltd. (Hangzhou, China). All other solvents and chemicals used were of analytical

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grade.

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Antibody Production

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Antigens Preparation. To prepare the artificial antigen, a novel derivatization

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method was used to modify the hapten of AFB1, by coupling it with a carrier protein

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via the active ester synthesis method.20 In briefly, 1 g of GA (C2H4O3) was first

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dissolved in 5 mL of TFA to produce solution A, whereas 12 mg of AFB1 was

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dissolved in 4 mL of acetonitrile (C2H3N) to obtain solution B. Then, solution B was

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added dropwise to solution A. The conjugation mixture was further stirred at room 5



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temperature (RT) for 20 min and then separated by liquid chromatography to obtain

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AFB1-GA. Then, 3 mg of AFB1-GA, 2 mg of NHS, and 3 mg of EDC were

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sequentially dissolved in 100 µL of DMF. After mixing at RT overnight, the activated

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hapten solution was added dropwise to 1 mL of a stirred solution of 9.4 mg of BSA.

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Finally, the AFB1-GA-BSA solution was stored at –20 °C for use as the immunogen.

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Determination of the efficiency of AFB1-GA conjugation to the carrier molecule was

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carried out by UV spectroscopy. AFB1-GA-OVA was also prepared by the active ester

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synthesis method for use as the coating antigen.

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IgY Generation. Five 150-day-old chickens were immunized with 400 µg of

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AFB1-GA-BSA in phosphate-buffered saline (PBS, pH 7.4) with an equal volume of

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FCA, at four different sites of the pectoral muscle. Four booster injections of the same

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amount of immunogen emulsified with FIA were administered at intervals of 3 weeks

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after the initial injection. The eggs were harvested after the initial immunization and

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stored at 4 °C before the extraction of the antibodies. The anti-AFB1 IgY (hereafter

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abbreviated as IgY) was purified by the ammonium sulfate precipitation method

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under acidic conditions.21 The IgY purity was identified by SDS-PAGE and quantified

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with ImageJ software.

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Competitive Indirect ELISA

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The competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was

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performed according to an ELISA protocol described previously.22 The 96-well plates

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were coated with AFB1- GA-OVA (100 µL/well), incubated overnight at 37 °C, and

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then washed twice with PBST solution containing 0.5% Tween-20 in PBS. The plates 6


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were then blocked with blocking buffer at 37 °C for 3 h and dried at 37 °C for 1 h. For

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the negative control wells of the pre-coated plates, IgY dilutions (50 µL/well) and

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PBS (50 µL/well) were added. For the experimental wells, PBS was replaced with 50

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µL of AFB1 standard. The plates were incubated at 37 °C for 40 min and then washed

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five times with PBST. After that, 100 µL of HRP-conjugated goat anti-chicken IgY

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(1:5000 in PBST) was added to each well and the plates were incubated at 37 °C for

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30 min. TMB solution (100 µL/well) was then added and the plates were incubated

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for 10 min. Finally, the reaction was stopped by the addition of 50 µL of 2 M H2SO4.

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The absorbance of the reaction solution at 450 nm (A450) was recorded. The

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four-parameter logistic function, plotted by Origin 8.5 (Origin Lab Corp.,

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Northampton, MA, USA), was fitted to establish the calibration curves. The IC50

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value was defined as the inhibitory concentration at 50% binding. The inhibition rate

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(I) was calculated using Eq. (1):

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I = [(A0-A)/A0] × 100%

(1)

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Where A0 and A represented the absorbance values of the negative well and AFB1

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standard well, respectively.

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Cell Viability

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L-02 and Swan 71 cells were incubated with different concentrations of AFB1

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and IgY for 12, 24, or 48 h. IgY was serially diluted in RPMI-1640 medium and each

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dilution was then preincubated for 30 min at 37 °C with an equal volume of AFB1 to

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obtain various IgY and AFB1 mixtures. For the toxin control, AFB1 in RPMI-1640

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medium without antibody was prepared. The IgY and AFB1 mixtures were added to 7



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respective L-02 cells monolayers (100 µL/well) and incubated for 48 h at 37 °C under

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5% CO2; anti-ZEN IgY and BSA were used as the negative control. IgY and AFB1

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mixtures and AFB1 alone were also added to Swan 71 cells monolayers, respectively.

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The cell viability was assessed by

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The A450

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value of the cells was determined using a microplate reader (Thermo Fisher Scientific,

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Waltham, MA, USA).

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Cell Cycle and Apoptosis

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L-02 cells were seeded in 6-well plates at 2 × 105 cells/well and divided into six

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groups according to varying AFB1 and IgY contents (i.e., 100 µM AFB1 alone, 0.5

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µM IgY alone, 100 µM AFB1 + 0.5 µM IgY, 100 µM AFB1 + 0.25 µM IgY, 100 µM

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AFB1 + 0.125 µM IgY, and 0.1% DMSO as a control). The IgY and AFB1 mixtures

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were added accordingly to the wells and incubated for 48 h. The cell cycle and

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apoptosis analyses were conducted using cell cycle and apoptosis detection kits

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according to the manufacturer’s instructions. The data were analyzed using FlowJo

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software (TreeStar, San Carlos, CA, USA).

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Cell Migration and Invasion

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Swan 71 cells were exposed to IgY and AFB1 mixtures for 48 h. The effect of the

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IgY and AFB1 mixtures on the migration and invasion of the cells was then assessed

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by trans-well assay.23 The cells were imaged under a BX51 fluorescence microscope

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(Olympus, Tokyo, Japan).

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ROS Production 8


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Cellular ROS production was measured using a ROS assay kit according to the

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manufacturer’s instructions. After treatment with the IgY and AFB1 mixtures as

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described above, the L-02 cells were collected and incubated with 10 µM

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2′,7′-dichlorofluorescein diacetate for 30 min at 37 °C. The 2′,7′-dichlorofluorescein

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in the cells was detected with a FC500 flow cytometer (Beckman Coulter Inc.,

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Minneapolis, MN, USA). ROS production was expressed as a fold change of the

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fluorescence intensity of the treated samples relative to the appropriate controls (fold

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increase over control).

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Mitochondrial Membrane Potential

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The MMP was monitored by determining the relative amounts of emissions

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signal from mitochondrial JC-1 monomers and aggregates. Mitochondrial

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depolarization was indicated by the increase of the green/red fluorescence intensity

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ratio. Cells were exposed to the IgY and AFB1 mixtures, followed by incubation with

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JC-1 staining solution (5 µg/mL) at 37 °C for 20 min, and were then washed twice

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with JC-1 staining buffer. All samples were observed by BX51 fluorescence

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microscopy (Olympus, Tokyo, Japan) to confirm the JC-1labeling patterns.

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Nuclear Morphology

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Nuclear changes and apoptotic body formation were detected by Hoechst 33342

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staining. L-02 cells were first treated with the indicated IgY and AFB1 mixtures. Then,

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the attached cells were treated with Hoechst 33342 (1×) at 37 °C for 10 min, washed

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twice with PBS, and shielded from light. The Hoechst-stained nuclei were observed

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under a BX51 fluorescence microscope (Olympus, Tokyo, Japan). 9



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Intracellular AFB1-DNA Adducts

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After exposure to the IgY and AFB1 mixtures, L-02 cells were harvested at

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1,000g for 5 min at RT. After the removal of most of the supernatant, the cell pellet

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was resuspended in 10–50 µL of the residual liquid. PBS (200 µL) was then added to

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wash the cells, followed by centrifugation at 1,000g at 4 °C for 5 min. The cell

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amount was determined with an FC500 flow cytometer (Beckman Coulter Inc.,

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Minneapolis, MN, USA). DNA was extracted using DNAzol reagent according to the

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manufacturer’s protocol. The AFB1-DNA adduct content was quantified with an

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AFB1-DNA ELISA kit. The AFB1-DNA content per million cells was measured with a

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microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

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Western Blotting Analysis

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L-02 cells were treated with the IgY and AFB1 mixtures and the total proteins

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were then determined by BCA method. Equal amounts of the cellular proteins were

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separated on a 10–12% SDS-PAGE gel and the bands were then transferred onto an

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equilibrated polyvinylidene difluoride membrane (Amersham Biosciences,

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Buckinghamshire, UK). After blocking with 5% skimmed milk in TBST (20 mM

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Tris-HCl, 1500 mM NaCl, and 0.1% Tween 20) at RT for 1 h, the membrane was

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incubated overnight with primary antibodies in blocking solution at 4 °C. The primary

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antibodies were rabbit monoclonal anti-SOD1, anti-CAT, anti-BCL2, anti-survivin,

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anti-caspase-3, anti-BAX and anti-β-actin. The membrane was washed three times in

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TBST for 15 min and then incubated with the secondary antibody in blocking solution

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at RT for 1 h. The proteins were detected by an enhanced chemiluminescence 10


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(Amersham Corporation, Arlington Heights, IL, USA), visualized with an LAS-3000

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bioimaging analyzer (Fuji Film, Tokyo, Japan), and analyzed with ImageJ software.

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Statistical Analysis

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The experiments were repeated at least three times. Relative values were

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standardized by setting the control values to 1.0, and all individual experimental

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values were calculated as folds changes relative to the control values. The data from

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different treatments were subjected to one-way analysis of variance. Values of P

IgY Reduces AFB1-Induced Cytotoxicity, Cellular Dysfunction, and

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