Egg Yolk Immunoglobulin Supplementation Prevents Rat Liver from

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Food Safety and Toxicology

Egg yolk immunoglobulins supplementation prevents rat liver from aflatoxin B1-induced oxidative damage and genotoxicity Taotao Qiu, Xing Shen, Xiangmei Li, Yun Yun Gong, Zhongmin Zou, Chunhong Liu, Feng Ye, Chenyang Mi, Zhen-Lin Xu, Yuanming Sun, Jie Lin, Huidong Zhang, and Hongtao Lei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04659 • Publication Date (Web): 17 Nov 2018 Downloaded from http://pubs.acs.org on November 18, 2018

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Journal of Agricultural and Food Chemistry

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Egg yolk immunoglobulins supplementation prevents rat liver from

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aflatoxin B1-induced oxidative damage and genotoxicity

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Taotao Qiu†, §, Xing Shen†, Xiangmei Li†, Yunyun Gong⊥, Zhongmin Zou ‡ , Chunhong

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Liu†, Feng Ye‡ , Chenyang Mi§, Zhenlin Xu†, Yuanming Sun†, Jie Lin†, Huidong Zhang*, §

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and Hongtao Lei *, †

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† Guangdong

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Provincial Key Laboratory of Food Quality and Safety / College of Food Science, South

China Agricultural University, Guangzhou, 510642, China. §Key

Laboratory of Environment and Female Reproductive Health / Public Health Laboratory Sciences

and Toxicology, West China School of Public Health, Sichuan University, Chengdu, China. ⊥ Department

of Food Safety Risk Assessment, China National Center for Food Safety Risk

Assessment, Ministry of Health, Beijing, 100021 PR China ‡ Institute

of Toxicology, College of Preventive Medicine, Third Military Medical University,

Chongqing, PR China

14 15

* E-mails: [email protected] (Lei H.), Tel.: +8620 8528 3925;

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[email protected] (Zhang H.), Tel.: +8623 6875 2292.

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ACS Paragon Plus Environment


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Abstract

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Egg yolk immunoglobulins (IgY), as nutraceutical supplement for therapeutic or

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prophylactic intervention, has been extensively studied. The effects of IgY on small

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molecular toxin-induced toxicity in animals is unclear. In the present study, the

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protection of highly purified and specific anti-AFB1 IgY against AFB1-induced

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genotoxicity and oxidative damage on the rat liver model were investigated. Our

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results revealed that AFB1 induced significant oxidative damage markers, as well as

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AFB1-induced protein expression in antioxidant, pro- and anti-apoptosis processes in

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rat liver. These effects could be significantly inhibited by co-gavage with anti-AFB1

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IgY in a dose-dependent manner. However, anti-AFB1 IgY did not significantly

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induce hepatic CAT and SOD1. To explore mechanisms, metabolite experiments were

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established to evaluate the influence of anti-AFB1 IgY on the absorption of AFB1 in

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rats. Middle and high dose of anti-AFB1 IgY reduced hepatic AFB1-DNA adducts by

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43.3% and 52.9%, AFB1-N7-guanine urinary adducts by 19.6% and 34.4%, and

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AFB1-albumin adducts by 10.5% and 21.1%, respectively. The feces of high dose

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anti-AFB1 IgY co-gavaged rats contained approximately 2-fold higher AFB1

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equivalents at 3–6 h after ingestion than AFB1 group feces, indicating IgY inhibited

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AFB1 uptake. These results had provided insight that anti-AFB1 IgY could prevent

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animal organs from damage caused by AFB1, and will be beneficial for the

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application of detoxication antibody as a supplement in food.

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Keywords: Anti-AFB1 IgY; Aflatoxin B1; Genotoxicity; Oxidative damage

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INTRODUCTION Chronic dietary aflatoxin B1 (AFB1) exposure is the main risk factors for

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hepatocellular carcinoma in various areas of Africa and south-east Asia. 1 AFB1 is

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metabolized by specific P450 enzymes to produce genotoxic metabolite

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(exo-8,9-epoxide AFB1, exo-AFBO), which binds nuclear DNA to form AFB1-DNA

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adducts. 2 AFB1-DNA adducts may introduce GC-TA transversion and block the

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normal replication and transcription, ultimately lead to DNA mutations and genomic

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instability causing genetic damage and transformation of hepatocytes. 3 Potential

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mechanism of hepatotoxicity induced by AFB1 is still under investigation. It is

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generally accepted that AFB1 can elevate level of reactive oxygen species (ROS) in

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the liver, which is considered to be one approach that leads to the subsequent

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hepatotoxicity. 4, 5 Nowdays, a lot of the research focused on nutritional supplements

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which may contribute to eliminate the harmful effects of environmental toxins and

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prevent multiple diseases in humans. 6 Nutritional supplements may be effective and

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safety approach to reduce the toxicity of mycotoxins in contaminated foods.

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Egg yolk immunoglobulins (IgY) are major antibody found in chicken egg yolk,

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and has been recognized as a lower cost and convenient source of polyclonal

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antibody. In recent years, there had been increasing interest in the protective role of

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specific IgY, which could highly effectively protect against a variety of intestinal

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pathogens 7 and macromolecular protein toxins (e.g., Ricin toxin). 8 IgY is a valuable

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nutraceutical and health supplement for prophylactic or therapeutic intervention via

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oral administration of mono-specific or mixed IgY formulations. 9 Orally ingested IgY 3



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was still immunologically active after passing through stomach, small intestine, and

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ileum. 10 IgY appears to be effectively resistant to chymotrypsin and trypsin

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degradation, has some advantages for oral administration, 11 and not be available for

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absorption of intact antibodies in humans. 12 Moreover, orally ingested IgY appears to

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have considerable potential to control diseases due to its high specificity. 13 Passive

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oral administration of IgY may be an emerging and promising nutritional strategy to

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reduce the risk of AFB1-contaminated foods.

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However, the effects of IgY in reducing small molecular toxin-induced toxicity in

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animals are unclear. In this study, we separated and purified IgY from egg yolk of hen

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that have been immunized with AFB1-GA-BSA. After evaluation of the binding

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activity of anti-AFB1 IgY to AFB1 in vitro, the effects of anti-AFB1 IgY and

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underlying the potential mechanisms in AFB1-induced oxidative damage and

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genotoxicity were investigated in rats. The female Sprague-Dawley (SD) rat gavage

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model of AFB1 was used to compare the effects of anti-AFB1 IgY co-gavage on

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AFB1-induced hepatic oxidative damage, and the effects of anti-AFB1 IgY

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co-treatment suppress early biomarkers of genetic damage including serum

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AFB1-albumin (AFB1-alb) adducts, liver AFB1-DNA adducts and urinary

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8,9-dihydro-8-(N7guanyl)-9-hydroxy AFB1 (AFB1-N7-Gua) excretion were also

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investigated.

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

MATERIALS AND METHODS

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Chemicals and Reagents

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Aflatoxin B1 was obtained from J&K Scientific (Beijing, China). Malondialdehyde 4


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(MDA) detection kit and nitric oxide (NO) detection kit were from Beyotime

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(Shanghai, China). Antibodies against catalase (CAT), superoxide dismutase 1

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(SOD1), and apoptosis regulator (survivin and activated-caspase-3) were all from

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Bioworld Technology, Inc. (St. Louis Park, MN, USA). AFB1-alb, AFB1-N7-Gua,

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AFB1-DNA, Aflatoxin M1 (AFM1) and Aflatoxin P1 (AFP1) enzyme-linked

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immunosorbent assay (ELISA) kits were obtained from Sinogeneclon Co., Ltd

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(Hangzhou, China). Anti-Zearalenone (ZEN) IgY was procured from Guangdong

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

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Production of Anti-AFB1 IgY

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The artificial antigen (AFB1-GA-BSA) was generated as described by the previous

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study. 14 Hens were immunized with 400 μg of AFB1-GA-BSA in PBS with an equal

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volume of Freund’s complete adjuvant for the first immunization or Freund’s

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incomplete adjuvant for four booster immunization. The anti-AFB1 IgY from egg yolk

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was purified by a combination of several purification technologies including

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water-soluble fraction under acidic conditions, filtration and ammonium sulfate

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precipitation method. 15 The IgY purity was analyzed by SDS-PAGE.

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Experimental animals

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For the pathophysiological study, three-week-old female SD rats (60–100 g) were

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housed three per cage in polycarbonate cages. For the metabolites study,

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four-week-old female SD rats (100–120 g) were housed individually in metabolic

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cages, for separate collection of feces and urines, with temperature at (22 ± 1 °C)

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under a 12 h light-dark cycle. All rats were maintained on standard lab diet. All 5



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animal experiments were conducted according to the ethical guidelines of the Animal

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Care and Use Committee of South China Agricultural University (Guangzhou, China,

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SYXK (Yue) 2014-0136).

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AFB1 binding with anti-AFB1 IgY in vitro

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Formation of a non-covalent complex between anti-AFB1 IgY and AFB1 was

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assessed by changing of AFB1 fluorescence. It provided sensitivity for monitoring

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fluorescence change with a microplate fluorescence reader (Thermo Fisher Scientific,

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Waltham, MA, USA). The initial concentration of AFB1 was 2.5 μM in 96-well

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plates. Anti-AFB1 IgY or anti-ZEN IgY was added in 0.25 nM increments up to 1

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μM, with negligible increase in assay volume from antibodies additions. The changes

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of fluorescence were detected at 442 nm emission with excitation at 360 nm.

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Pathophysiological experimental design

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Fifty-Four female SD rats were randomly divided into six treatment groups as

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shown in Table S1. Groups 1–3 (12 rats/group) were experimental groups, and groups

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4–6 (6 rats/group) were controls groups. Rats in group 1 were received 200 μg/kg·BW

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AFB1 alone. Rats in group 2 were received 200 μg/kg·BW AFB1 mixed with 1.4

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mg/kg·BW anti-AFB1 IgY. Rats in group 3 were received 200 μg/kg·BW AFB1

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mixed with 0.09 mg/kg anti-AFB1 IgY. Rats in group 4 were received 200 μg/kg·BW

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AFB1 mixed with 1.4 mg/kg·BW anti-ZEN IgY. Rats in group 5 and group 6 were

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received 1.4 mg/kg·BW anti-AFB1 IgY and vehicle, respectively. The dose of AFB1

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and anti-AFB1 IgY administered were based on the results of binding activity study.

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All groups were orally administered five times per week for 4 weeks. Rats were 6


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carefully weighed two times a week throughout the treatment period. Blood samples

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were collected from all animals for the determination of aspartate amin otransferase

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(AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), indirect

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bilirubin (IB), and total bilirubin (TB) at the end of the treatment period. Serum

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biochemical parameters including AST, ALT, ALP, IB, and TB were determined

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according to the manufacturer’s instructions. Liver tissue samples were taken from

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rats within the different treatment groups.

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Tissue samples from the liver of each rat were divided into three parts. The first

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liver tissue sample was soaked in 10% buffered formalin for 24 h, hydrated and

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cleared in xylene, embedded in paraffin wax, sliced, and stained for histopathological

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examination using a BX51 fluorescence microscope (Olympus, Tokyo, Japan). The

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second liver tissue sample was used for NO and MDA measurement. The third liver

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tissue sample was immediately frozen at -80 oC for protein expression of antioxidation

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and apoptosis.

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NO, and lipid peroxidation measurement

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Liver tissues samples (0.05 ± 0.01 g) were homogenized

in PBS (20% w/v). This

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homogenate was subjected to centrifugation at 12000 g and 4 °C for 10 min and the

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supernatants was collected for NO and MDA measurement. The total NO assay kit

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and the MDA detection kit were selected for NO and MDA measurement,

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respectively. Assays for NO and MDA were performed according to the

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manufacturer’s protocol. The protein content in the supernatant of each rat liver was

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measured by BCA method. 7



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Western Blotting

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Samples of liver tissues were lysed via ultrasound at 4 °C in radio

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immunoprecipitation assay buffer with protease inhibitors. Proteins content was

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determined by BCA method. Samples containing equal amount of proteins were

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separated by 12% SDS-PAGE gel, and then the proteins were transferred onto

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equilibrated polyvinylidene difluoride membrane (Millipore Corp, Bedford, MA,

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USA). After blocking with 5% non-fat milk in TBST (20 mM Tris-HCl, 1500 mM

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NaCl, and 0.1% Tween 20) at room temperature (RT) for 1 h, the membrane was

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incubated at 4 °C overnight with primary antibodies (anti-SOD1, anti-CAT,

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anti-survivin, anti-activated-caspase-3, and anti-β-actin). After washing in TBST for

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15 min, the membrane was incubated with the secondary antibody for 1 h, and

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detected by enhanced chemiluminescence (Amersham Corporation, Arlington

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Heights, IL, USA). The individual band intensities were analyzed by ImageJ software.

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Metabolites experimental design

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Female SD rats were randomly arranged into six treatment groups as shown in

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Table S1. Rats in group 1 (6 rats/group) were received 400 μg/kg·BW AFB1. Rats in

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group 2 (6 rats/group) were received 400 μg/kg·BW AFB1 mixed with 2.8 mg/kg·BW

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anti-AFB1 IgY. Rats in group 3 (6 rats/group) were received 400 μg/kg·BW AFB1

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mixed with 0.72 mg/kg·BW anti-AFB1 IgY. Rats in group 4 (6 rats/group) were

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received 400 μg/kg·BW AFB1 mixed with 0.18 mg/kg·BW anti-AFB1 IgY. Rats in

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group 5 (6 rats/group) and group 6 (6 rats/group) were received 2.8 mg/kg·BW

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anti-AFB1 IgY and vehicle, respectively. All rats were treated on days 0, 1, and 2. The 8


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treated rats were sacrificed 2 h after final treatment. Liver and blood samples were

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collected from all animals within different treatment groups to quantify AFB1-DNA

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adducts and AFB1-alb adducts. Fecal samples and urine were collected to quantify

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AFB1 and metabolites (AFP1, AFM1 and AFB1-N7-Gua).

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Quantification of AFB1 in feces

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Every 3 hours fecal samples were collected after day 0 administration for 24 h, and

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then dried, weighed and ground to powder. Fecal powder sample (200 mg) was

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homogenized in 1 mL water. 2 mL dichloromethane was added to the sample. Fecal

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samples were centrifuged at 5000 g for 10 min at RT to obtain supernatant. Finally,

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the AFB1 were identified and quantified by HLPC as described. 16 A 20 μL sample

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was analyzed using LC-20A Prominence HPLC system (Shimadzu, Kyoto, Japan), a

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fluorescence detector at an emission wavelength of 440 nm and an excitation

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wavelength of 360 nm. The mobile phase was 45% methanol filtered through a 0.45

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μm filter, and flow rate at 0.7 mL min-1 flow at 28 °C with Waters C18 XBridge

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columns. AFB1 concentration was determined in all of the fecal samples.

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Quantification of AFB1-alb, AFB1-DNA, and AFB1-N7-Gua

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Plasma and serum of blood samples were centrifuged at 4,000 g at 4 °C for 10 min

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and the resultant serum was removed for the determination of AFB1-alb levels. Liver

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tissues samples (0.05 ± 0.01 g) was weighed and homogenized in PBS, and then DNA

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was extracted using DNAzol reagent according to the kits protocol for the

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determination of AFB1-DNA levels. Twenty-four hour urine samples were normalized

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using a spectrophotometric creatinine kit. The content of AFB1-alb in blood, 9



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AFB1-DNA adducts in liver, and metabolites (AFP1, AFM1 and AFB1-N7-Gua) in

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urine were quantified by ELISA method.

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Statistical analysis

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All data are expressed as means ± SEM. Shapiro-Wilk test was used to identify

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data normality. Non-parametric tests were used to compare the data that were not

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normally distributed. The differences between the two groups were determined by

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Student's t-test. Significance was based on probability of P < 0.05.

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

RESULTS

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Purified Anti-AFB1 IgY complexed with AFB1 in vitro

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Anti-AFB1 IgY purity was identified by SDS-PAGE. As shown in Fig. S1A, two

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bands were observed on the gel, corresponding to the 25 kDa light chain and the 65

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kDa heavy chain of IgY, respectively. 17 We have previously reported that anti-AFB1

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IgY formed a strong non-covalent complex with AFB1 by indirect competitive

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ELISA. 14 To evaluate the ability of anti-AFB1 IgY to complex with AFB1, AFB1 (2.5

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μM) and different concentrations of anti-AFB1 IgY were mixed in vitro, and the

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fluorescent intensity was determined by a fluorescent microplate reader in change of

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the AFB1 fluorescence spectrum from 380 to 550 nm. There was appropriate linear of

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fluorescence at low concentrations of AFB1, as shown in Fig. S2A. Fluorescence of

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different concentrations (0–2 μM) anti-AFB1 IgY and anti-ZEN IgY were weak (Fig.

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S2B). To quantify the change in the fluorescent intensity of AFB1 with anti-AFB1

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IgY, the fractional fluorescence changes at 442 nm emission with each titration was

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plotted and a model assuming a 80 : 1 ratio of AFB1 to anti-AFB1 IgY binding (Fig. 10


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S1B). As control, anti-ZEN IgY showed no obvious change of fluorescent value of

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AFB1 (Fig. S1B).

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Body weight effect of anti-AFB1 IgY on AFB1-cogavaged rat

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The body weight gain rate in rats of different treatments groups revealed that

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treatment with high dose anti-AFB1 IgY alone had no significant differences on

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growth relative to treatment with vehicle throughout the experimental period. By the

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eleventh day of the experiment, pathophysiological experimental rats had attained the

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same weight growth rate, and rats treated with AFB1 alone showed slower growth rate

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compared with the other groups rats later. Rats co-gavaged with AFB1 and the high

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dose of anti-AFB1 IgY showed a significant increase in body weight gain rate relative

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to rats treated with AFB1 alone. Rats receiving the combined treatment of AFB1 with

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the low dose of anti-AFB1 IgY or anti-ZEN IgY had no significant differences on

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growth compared with rats receiving AFB1 only (Fig. S3).

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Effect of anti-AFB1 IgY on liver function parameters on AFB1-cogavaged rat

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The liver function parameters in different treatments groups are presented in Table

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1. Rats treated with AFB1 induced liver damage as indicated by the significant

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increases in AST, ALT, ALP, IB, and TB; anti-AFB1 IgY alone showed insignificant

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changes in AST, ALT, ALP, IB, and TB. Rats co-gavaged with the low dose of

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anti-AFB1 IgY had significant decrease in IB, TB, and AST relative to rats treated

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with AFB1 alone; ALT, AST, and ALP showed no changes. Rats co-gavaged with the

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high dose of anti-AFB1 IgY showed a significant decrease in ALT, AST, ALP, IB,

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and TB compared with the AFB1 group. In contrast, treatment with AFB1 co-gavaged 11



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with anti-ZEN IgY failed to induce any improvement in all biochemical parameters

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compared with the AFB1 group.

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Anti-AFB1 IgY improved AFB1-induced lipid peroxidation in the liver

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Rats treated with AFB1 showed a significant increase in the levels of MDA and NO

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in the liver tissue compared with the control group (Table 2). However, MDA and NO

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levels showed a significant decrease in response to anti-AFB1 IgY co-gavage

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compared with the AFB1 group. Meanwhile, high-dose anti-AFB1 IgY co-gavage was

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more effective than low-dose anti-AFB1 IgY. Administration of anti-AFB1 IgY did

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not significantly increased MDA and NO generation in the liver tissue compared with

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the control group. Anti-ZEN IgY co-gavage resulted no influence in MDA and NO

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comparison to the AFB1 group.

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Anti-AFB1 IgY attenuates hepatic pathology induced by AFB1 in rat

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Liver sections of control or anti-AFB1 IgY group showed no visible lesions and

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central veins surrounded by polygonal cells (Fig. 1A and B). The liver sections of

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AFB1 group showed significant histopathological lesions, which were characterized

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by swelling and a vacuolar appearance of hepatocytes. Livers from the AFB1 group

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also showed lymphocyte infiltration in hepatocytes and bile duct epithelium

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hyperplasia (Fig. 1C). Livers from rats co-gavaged with AFB1 and anti-AFB1 IgY

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showed a slight lesion (Fig. 1E and F). In contrast, treatment with AFB1 co-gavaged

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with anti-ZEN IgY failed to attenuate hepatic pathology induced by AFB1 in rat (Fig.

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1D).

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Expression of antioxidation and apoptosis protein in the liver 12


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The relative abundance of antioxidant and apoptosis proteins, including SOD1,

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CAT, activated-caspase-3, and survivin in liver were examined by Western blotting.

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There was no significant difference in the expression of antioxidant and apoptosis

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proteins between the anti-AFB1 IgY group and the control group. The results showed

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a significant increase in the expression of activated-caspase-3 accompanied with a

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significant decrease in the expression of survivin in livers from rats with AFB1 alone.

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However, co-treatment with high dose of anti-AFB1 IgY resulted in significant

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changes in the expression levels of these apoptotic proteins (Fig. 2A, B). The results

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of Western blotting were well consistent with the results of histopathological analysis.

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Fig. 2C and D shows the activity levels of CAT and SOD1 on day 60 in each

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treatment, which indicated evidence of decreased CAT and SOD1 activity in AFB1

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group relative to control on day 60. Co-treatment with anti-AFB1 IgY significantly

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promoted the CAT and SOD1 expression in liver compared with the AFB1 group.

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Moreover, treatment with anti-AFB1 IgY alone did not induce CAT and SOD1.

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Effect of anti-AFB1 IgY on levels of AFB1 metabolites in feces and urine

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The absorption of AFB1 in the rat gut within a prescribed time limit was examined

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by analyzing feces collected at 3 hour intervals. The anti-AFB1 IgY co-gavaged group

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showed an overall higher level of AFB1 at five different time periods, relative to the

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AFB1 group. The anti-AFB1 IgY co-gavaged group showed no influence on AFB1

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excretion time, and 3–6 h after first gavaged AFB1 level was at the maximum. On day

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1, 3–6 h feces from the 0.72 and 2.8 mg/kg·BW anti-AFB1 IgY co-gavaged rats

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contained 149.6% and 180.7% more AFB1 equivalents than the AFB1 rats, 13



280 281

respectively (Fig. 3A). The mean levels of two major less-toxic AFB1 metabolites (AFM1 and AFP1) and

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AFB1-N7-Gua in 24 h urine samples taken on day 0 are showed in Fig. 4A–C.

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Conversely, both 0.72 and 2.8 mg/kg·BW anti-AFB1 IgY co-treatments significantly

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reduced the level of the AFB1-N7-Gua adduct excreted in the urine by 19.6% and

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34.4% as compared with the AFB1 group, respectively. AFM1 in the urine was

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reduced by 9.1% and 29.0%, respectively. AFP1 excretion in the urine was reduced by

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16.7% and 26.1%, respectively. The relative amounts of protection by anti-AFB1 IgY

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against the urinary AFB1-DNA repair product AFB1-N7-Gua and AFB1 metabolites,

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AFM1 and AFP1, were identical.

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Effect of anti-AFB1 IgY on AFB1-alb in serum

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AFB1 group showed a higher level of serum albumin adducts compared with

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control. The level of the AFB1-alb adduct in serum was significantly reduced by

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10.5% and 21% by co-gavage with 0.72 and 2.8 mg/kg·BW anti-AFB1 IgY,

294

respectively, compared with the AFB1 group (Fig. 3B).

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Effect of anti-AFB1 IgY on AFB1-DNA adduct in the liver

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The effects of anti-AFB1 IgY on hepatic AFB1-DNA adducts in rats gavaged with

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400 mg/kg·BW AFB1 for 3 days are shown in Fig. 3C. Liver samples were taken at 2

298

h after final treatment. This is consistent with previous studies in which the maximum

299

levels of AFB1-DNA adducts are observed following the first treatment with AFB1. 18

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Co-gavage with 0.72 and 2.8 mg/kg·BW anti-AFB1 IgY reduced hepatic AFB1-DNA

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adducts by 43.3% and 52.9%, respectively, compared with the AFB1 group. 14


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

DISCUSSION

In the present work, high specificity anti-AFB1 IgY was characterized in vitro, and

304

then the ability of the anti-AFB1 IgY to protect rat liver against AFB1 damage was

305

evaluated by monitoring the effects on metabolite oxidative stress, blood biochemical

306

analysis, hepatic damage, and protein expression of liver cells. The results of the

307

blood biochemical and metabolite oxidative stress analysis showed anti-AFB1 IgY

308

could alleviate hepatic damage by AFB1 in an anti-AFB1 IgY dose-dependent manner.

309

These results were supported by the histopathological examination of liver sections.

310

To determine the impact of the co-treatment on biomarkers of AFB1 damage a

311

number of biomarkers were examined. Urinary AFB1-N7-Gua, AFM1, and AFP1 as

312

well as liver AFB1-DNA adducts and serum AFB1-alb adducts levels were

313

dramatically reduced in anti-AFB1 IgY co-gavaged rats compared with the

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AFB1-treated group. We also found that more AFB1 equivalents was excreted in the

315

anti-AFB1 IgY co-exposed group, suggesting that the antibody reduced uptake of

316

AFB1. Taken together, these results indicate that hepatic damage by AFB1 can be

317

alleviated in rats by co-treatment with anti-AFB1 IgY.

318

A great deal of evidence has demonstrated that aflatoxins are metabolized by the

319

liver-specific cytochrome P450 enzyme system to produce a highly reactive

320

intermediate, exo-AFBO, which binds covalently to DNA and forms the major

321

AFB1-N7-Gua adducts. 19 The formation of AFB1-DNA adducts is regarded as an

322

important mechanism in the initiation of AFB1-induced hepatocarcinogenesis. 20

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These genotoxic endpoints, AFB1-DNA and AFB1-N7-Gua, are well known markers 15



324

of genotoxicity. Many studies have reported that any reduction in the frequency of

325

these genotoxic endpoints gives an indication of the antigenotoxicity of a particular

326

compound. 21 In this study, treatment with AFB1 induced AFB1-N7-Gua in urine and

327

AFB1-DNA in liver relative to the control rats in which no AFB1-DNA were

328

observed. The reduction of these biomarkers by co-administration of specific

329

anti-AFB1 IgY shows the potential to prevent the development of carcinogenesis and

330

other liver damage due to AFB1. For example, dietary chlorophyll could attenuate the

331

degree of AFB1-DNA adducts in rats, which is correlated with reduced incidence of

332

pre-neoplastic lesions. 22 This level of protection was mirrored by commensurate

333

decreases AFB1-alb in serum, AFB1-N7-Gua in urinary, and increases AFB1

334

equivalents in feces excretion. 23 Similarly, our results showed that co-gavage with

335

anti-AFB1 IgY succeeded to reduce AFB1-alb in serum, AFB1-DNA adducts in liver,

336

AFB1-N7-Gua in urine and increases AFB1 in the feces of rats treated AFB1.

337

Meanwhile, anti-AFB1 IgY co-treatment resulted in significant changes in the

338

expression levels of apoptotic proteins induced by AFB1. Hence, the present study

339

demonstrated that anti-AFB1 IgY co-treatment protects against AFB1-induced damage

340

in liver.

341

The AFB1 toxicity may also be attributed to oxidative stress. AFB1 induces

342

important liver damage, as shown by the significant increase in ROS formation in rat

343

liver as indicated by the significant increment of MDA and NO, 24, 25 accompanied

344

with a remarkable change in total antioxidant capacity in rats such as CAT, SOD1 and

345

GPx. 26 The oxidative stress may be one of the underlining mechanisms for 16


Page 16 of 36

Page 17 of 36


346

AFB1-induced tumorigenesis. 27, 28 The results of this study strongly support previous

347

reports which suggested that AFB1 or its metabolites may lead to oxidative stress. 29

348

One way to ameliorate the effects of AFB1 is via increase in the activity of antioxidant

349

enzymes. 30 However, our results suggest that anti-AFB1 IgY does not exert a

350

protective effect by eliciting antioxidation enzyme in detoxification processes of

351

AFB1 when co-treatment with the AFB1. Similar to our results, Zeynalian reported

352

there was no difference in the MDA level and the activity of CAT, GPx and SOD1

353

between the IgY group and the control group. 31 The results of this work was

354

consistent with previous studies, which the hepatic CAT and SOD1 in rats treated

355

with anti-AFB1 IgY alone have no increase in these antioxidation activities compared

356

with the control livers. Data on the metabolism of IgY in the whole rat gut as well as

357

uptake of IgY from the gut are very limited, but for decades the central assumption

358

has been that IgY uptake in vivo is not significant. Such poor uptake is a likely

359

explanation for the lack of effect on the antioxidant enzymes in our study.

360

It is now widely accepted that polyclonal native IgY obtained from egg yolk after

361

immunization of hens is emerging as specific targeting agent for the prevention and

362

passive immunotherapy of various human and animal infectious diseases. 32 Although

363

immunogenic when applied systemically, the oral administration of specific IgY

364

antibodies provided an available way for therapeutic interventions with respect to a

365

variety of pathologies. However, the present study provides limited evidence that

366

specific IgY inhibits uptake of small molecular toxins in animals. We found that the

367

adduct burden of AFB1 in rat tissues such as the liver, serum and urine, were reduced 17



368

over 10% to 53% range by anti-AFB1 IgY co- treatment, whereas in the feces of the

369

same rats, roughly 180% more AFB1 equivalents were excreted compared with the

370

AFB1 rats. Thus, anti-AFB1 IgY co-treatment largely restricted AFB1 to the

371

gastrointestinal absorption in rats. Moreover, substantial prevention was provided by

372

anti-AFB1 IgY against AFB1-induced hepatic oxidative damage. Our results suggest

373

that anti-AFB1 IgY prevents AFB1 uptake, thereby reducing the effects of the AFB1.

374

One simple mechanism to explain inhibition of uptake of the AFB1 is the formation of

375

a molecular complex between anti-AFB1 IgY and AFB1 in the gut. We showed that

376

anti-AFB1 IgY was able to form an AFB1 complex of 80 times greater stability in

377

vitro. IgY is highly specific for binding antigens. 33 The specificity of the prevention

378

of anti-AFB1 IgY was shown by the fact that an equimolar dose of anti-ZEN IgY did

379

not produce the prevention against AFB1-induced injury in liver afforded by the

380

anti-AFB1 IgY.

381

Additional studies about a quantitative relationship between the amount of complex

382

formation over a range of anti-AFB1 IgY administered dose and the resulting

383

AFB1-induced oxidative damage and genotoxicity in rat liver was carried out to

384

determine the relative importance of complex formation in prevention by anti-AFB1

385

IgY. Here, simultaneous administration of low-dose anti-AFB1 IgY showed relatively

386

weak effects.

387

In conclusion, anti-AFB1 IgY was a potent nutritional preventive agent against

388

early biochemical biomarkers of AFB1 damage in the rat liver. IgY-mediated

389

prevention of AFB1-induced liver damage showed no effect on inducing hepatic 18


Page 18 of 36

Page 19 of 36


390

antioxidant enzymes activities in the present study. Urinary, serum, and liver

391

elimination and fecal metabolite studies provide supporting evidence that anti-AFB1

392

IgY prevented rat liver from AFB1-induced oxidative damage and genotoxicity by

393

reducing the availability of AFB1 to the liver due to inhibiting AFB1 uptake from the

394

gut. Anti-AFB1 IgY was able to recognize and AFB1, and led to the formation of

395

non-covalent complexes, then may reflect poor uptake in vivo. This study is the first

396

to report prevention effect by oral IgY against small molecular toxins-induced injury

397

development in an animal model, with the prevention observed against AFB1 damage

398

in liver.

399 400



401

Supporting Information

402

Figure S1, SDS-PAGE analysis of anti-AFB1 IgY and quantification of IgY changing

403

fluorescent value of AFB1. Figure S2, Fluorescence of different concentrations AFB1,

404

anti-AFB1 IgY, and anti-ZEN IgY. Figure S3, Effect of supplement with anti-AFB1

405

IgY on body weight gain rate in AFB1-gavaged rat. Table S1, Experimental treatment,

406

gavage schedule and experimental time

407



408

Corresponding Author

409

*E-mail: [email protected]. Phone: +86 20-8528-3448. Fax: +86 20-8528-0270.

410

(Hongtao Lei)

411

*E-mails: [email protected]. Phone: +86 23-6875-2292. (Huidong Zhang)

ASSOCIATED CONTENT

AUTHOR INFORMATION

19



412

ORCID

413

Hongtao Lei: 0000-0002-1697-1747

414

Huidong Zhang: 0000-0001-9810-549 X

415

Funding This work was supported by the National Key Research and Development Program

416 417

of China (2017YFC1601700, 2017YFC1002002, 2017YFC1601205,

418

2016YFE0106000), and the Fundamental Research Funds for the Central

419

Universities, Natural Science Foundation of China (31370793, 81422041, 21475047,

420

31701703, 31601555, 71633002), and Guangdong and Guangzhou Planned Program

421

in Science and Technology (S2013030013338, 2017B020207010, 201604030004,

422

2014A030306026).

423 424

Notes

425

We thank Dr Michael N Routledge in School of Medicine, University of Leeds for

426

help with revising the manuscript and language assistance. The authors declare no

427

competing financial interest.

428 429



430

IgY, egg yolk immunoglobulins; AFB1, Aflatoxin B1; AFM1, Aflatoxin M1; AFP1,

431

Aflatoxin P1; ZEN, Zearalenone; ROS, reactive oxygen species; exo-AFBO, AFB1

432

exo-8,9-epoxide; AFB1-alb, AFB1-albumin; AFB1-N7-Gua,

433

9-dihydro-8-(N7guanyl)-9-hydroxyAFB1; SOD1, superoxide dismutase 1; CAT,

ABBREVIATIONS USED

20


Page 20 of 36

Page 21 of 36


434

catalase; GPx, Glutathione peroxidase; NO, nitric oxide; GA, glycollic acid; PBS,

435

phosphate-buffered saline; MDA, malondialdehyde; ALT, alanine aminotransferase;

436

ALP, alkaline phosphatase; AST, aspartate aminotransferase; TB, total bilirubin; IB,

437

indirect bilirubin; ELISA, enzyme-linked immunosorbent assay; RT, room

438

temperature.

439

21



440



441

(1) Kar, P. Risk factors for hepatocellular carcinoma in india. J. Clin. Exp. Hep. 2014,

442

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27



554

Figure legends

555

Figure 1 Hepatic pathology observations. Photomicrographs (optical microscopy) of

556

haematoxylin and eosin stained rats liver sections from different treatments. Rats were

557

treated with vehicle as the control (A), or 1.4 mg/kg·BW anti-AFB1 IgY (B), or 200

558

μg/kg·BW AFB1 alone (C), or 200 μg/kg·BW AFB1 + 1.4 mg/kg·BW anti-ZEN IgY

559

(D), or 200 μg/kg·BW AFB1 + 1.4 mg/kg·BW anti-AFB1 IgY (E), or 200 μg/kg·BW

560

AFB1 + 0.09 mg/kg anti-AFB1 IgY (F). Normal liver tissue with central veins

561

surrounding by polygonal cells arranged in regular cords in the control group (A) and

562

group (B). Obvious liver injury with swelling, bile duct epithelium hyperplasia and

563

lymphocyte infiltration in hepatocytes (black arrow) in group (C) and group (D).

564

Liver with minor injury have almost normal architecture in group (E) and group (F).

565

Original magnification: ×200

566 567

Figure 2 Western blotting analysis of apoptosis-related (A) activated-caspase-3, (B)

568

survivin, and antioxidant-related (C) SOD1 and (D) CAT protein in liver tissues of

569

control, anti-AFB1 IgY treated, AFB1 treated, and AFB1 + anti-AFB1 IgY treated rats.

570

Values are the mean ± SEM (n = 3 per group). (∗) p < 0.05, significantly different

571

from the AFB1-treated group.

572 573

Figure 3 Effect of anti-AFB1 IgY on fecal elimination of AFB1, AFB1-induced serum

574

albumin adducts and hepatic DNA adducts. (A) AFB1, (B) AFB1-alb adducts, and (C)

575

AFB1-DNA adducts were measured as described in materials and methods. Values are 28


Page 28 of 36

Page 29 of 36


576

the mean ± SEM for N=6 rats per group. (∗) p < 0.05, significantly different from the

577

AFB1-treated group.

578 579

Figure 4 Effect of anti-AFB1 IgY on AFB1 metabolites in rat urine. Twenty-four hour

580

urine samples collected after the day 0 gavage were analyzed as described in materials

581

and methods. (A) AFB1-N7-Gua, (B) AFM1, (C) AFP1. Values are the mean ± SEM

582

for N=6 rats per group. (∗) p < 0.05, significantly different from the AFB1-treated

583

group.

584

29



Page 30 of 36

585

Table 1 Effect of AFB1 and/or anti-AFB1 IgY on liver function in rats treated for 60

586

days. Group T1-Control

T1-AFB1 a)

T1-IgY

T1-ALI b)

T1-AHI c)

T1-AZI d)

35.3±4.40

30.73±1.65*

39.33±5.45

Parameter ALT (U/L)

27.47±2.74* 31.93±4.99* 41.30±2.34

AST (U/L)

100±15.30* 122.2±25.95* 191.23±19.02 145.2±12.44* 137.13±13.16* 180.73±27.13

ALP (U/L)

61.7±16.93* 72.33±8.62* 108±4.36

93.33±8.14

86.67±5.86*

103.33±17.21

TB (μmol/L) 1.53±0.06 * 1.63±0.21*

2.5±0.20

1.97±0.15*

1.63±0.31*

2.7±0.26

IB (μmol/L) 1.00±0.24*

2.15±0.13

1.73±0.14*

1.15±0.30*

1.93±0.17

1.40±0.22*

587

a) AFB1: 200 μg/kg·BW AFB1.

588

b) T1-ALI: 200 μg/kg·BW AFB1 + 0.09 mg/kg·BW anti-AFB1 IgY;

589

c) T1-AHI: 200 μg/kg·BW AFB1 + 1.4 mg/kg·BW anti-AFB1 IgY;

590

d) T1-AZI: 200 μg/kg·BW AFB1 + 1.4 mg/kg·BW anti-ZEN IgY;

591

Data are expressed as the mean ± SEM, n = 3. (∗) p < 0.05, significantly different

592


593

30


Page 31 of 36


594

Table 2 Effect of AFB1 and/or anti-AFB1 IgY for 60 days on levels of MDA and NO

595

in rat liver tissues. Group T1-Control

T1-IgY

T1-AFB1 a)

T1-ALI b)

T1-AHI c)

T1-AZI d)

Parameter MDA (nmol/mg protein) 14.42±0.81* 13.5±1.41* 25.49±1.50 17.58±1.55* 15.60±1.07* 22.78±1.40 NO (nmol/mg protein)

1.51±0.01*

1.74±0.38* 2.45±0.16

2.47±0.05

2.12±0.09* 2.48±0.05

596

a) AFB1: 200 μg/kg·BW AFB1.

597

b) T1-ALI: 200 μg/kg·BW AFB1 + 0.09 mg/kg·BW anti-AFB1 IgY;

598

c) T1-AHI: 200 μg/kg·BW AFB1 + 1.4 mg/kg·BW anti-AFB1 IgY;

599

d) T1-AZI: 200 μg/kg·BW AFB1 + 1.4 mg/kg·BW anti-ZEN IgY;

600

Data are expressed as the mean ± SEM, n = 3. (∗) p < 0.05, significantly different

601


602

31



603

Figure 1 A

B

C

D

E

F

604 605

32


Page 32 of 36

Page 33 of 36

606


Figure 2 Activated-caspase-3

A Activated-Caspase-3

*

β-actin

1.0

0.5

0.5

0.0

0.0

Relative ratio

C SOD1 CAT

D

SOD1 1.5

—

200

IgY (mg/kg·BW) —

1

—

200 200 0.9

1.4

*

1.0

0.5

0.0

AFB 1 (μg/kg·BW) IgY (mg/kg·BW)

CAT 1.5

*

1.0

0.5

β-actin AFB1 (μg/kg·BW) —

Survivin 1.5

*

1.0

Survivin

607

B

1.5

0.0

—

—

200

200

—

1

—

0.9

608

33


— 200 AFB 1 (μg/kg·BW) — 1.4 IgY (mg/kg·BW)

—

200

200

200

1

—

0.9

1.4


Figure 3

34


45

30

*

*

15

*

I H

M I

-A T2

LI -A

FB 1

-A T2

T2

T2

-A T2

-C

-Ig Y

0 on tr ol

ng AFB1 -DNA equivalents/g liver

AFB1-DNA

C

T2

I H -A

M I

24

-A

21

T2

18

LI

12 15 Time (h)

-A

9

T2

6

T2

3

4

FB 1

0 0

*

-A

200

8

-Ig Y

T2 -AHI

400

12

T2

T2 -AMI

T2

T2 -ALI

600

AFB1-alb

B

on tr ol

T2 -AFB1

nmol AFB1 -alb equivalents/g albumin

AFB1

*

-C

610 611

A

T2

Pmoles AFB1 equivalents/ g feces

609

Page 34 of 36

613 I

300

200

*

100

0

614

35


T2

H

M I

LI

-A

-A

-A

FB 1

I

-Ig Y

C

T2

-A

T2

on tr ol

Pmol AFP1 equivalents/moml creatinine

H

M I -A

-A

LI

FB 1

-Ig Y

-A

T2

T2

T2

-A

T2

on tr ol

AFM1

T2

0

B

T2

75

-C

*

T2

150

T2

*

Pmol AFM1 equivalents/moml creatinine

I

225

-C

H

M I

-A

-A

LI

FB 1

-Ig Y

-A

T2

T2

T2

-A

T2

AFB1-N7-guanine

T2

Pmol AFB1 -N7 -guanine/mmol creatinine

on tr ol

A

T2

-C

612

T2

Page 35 of 36 Journal of Agricultural and Food Chemistry

Figure 4 AFP1

45

30

*

15

0


615

TOC Graphic

616

36


Page 36 of 36

Egg Yolk Immunoglobulin Supplementation Prevents Rat Liver from

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