Food and Chemical Toxicology 59 (2013) 470–475

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

Effect of the combined probiotics with aflatoxin B1-degrading enzymeon aflatoxin detoxification, broiler production performance and hepaticenzyme gene expression

0278-6915/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2013.06.044

⇑ Corresponding author at: College of Animal Science and Veterinary Medicine,Henan Agricultural University, Zhengzhou 450002, China. Tel.: +86 371 63558180;fax: +86 371 63558998.

E-mail address: qqy1964@126.com (Q.-q. Yin).

Rui-yu Zuo a,b, Juan Chang a, Qing-qiang Yin a,c,⇑, Ping Wang a, Yu-rong Yang a, Xiao Wang c,Guo-qiang Wang d, Qiu-hong Zheng a

a College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Chinab Henan Husbandry Breau, Zhengzhou 450002, Chinac Henan Engineering and Technology Research Center of Feed Microbes, Zhoukou 466000, Chinad Nanyang Agricultural School, Nanyan 473000, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 April 2013Accepted 22 June 2013Available online 2 July 2013

Keywords:Aflatoxin B1

ProbioticsAFB1-degrading enzymeBroiler production performanceGene expressions

In order to degrade aflatoxin B1 (AFB1), AFBl-degrading microbes (probiotics) such as Lactobacillus casei,Bacillus subtilis and Pichia anomala, and the AFBl-degrading enzyme from Aspergillus oryzae were selectedand combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomlydivided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as thebasal diet, 400 lg/kg AFB1 supplement without feed additive, and 200, 400, 800 lg/kg AFB1 supplementwith 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine pro-duction performance of broilers. In addition, serum, liver and chest muscle were selected for measuringAFB1 residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB1 on chicken’s pro-duction performance and nutrient metabolic rates (P < 0.05). It could also improve AFB1 metabolism,hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxi-doreductase, apoptosis, cell growth, immune system and metabolic process (P < 0.05). It could be con-cluded that the feed additive was able to degrade AFB1 and improve animal production.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Aflatoxins are secondary metabolites produced by toxigenicstrains of Aspergillus flavus (A. flavus), Aspergillus nomius, Aspergillusparasiticus and so on, which are known to be potent carcinogensand hepatotoxic agents and pose serious hazards to human andanimal health (Sidhu et al., 2009). It is estimated that about 5 bil-lion people worldwide suffer from uncontrolled exposure to afla-toxins (Strosnider et al., 2006). In addition, aflatoxins can causeserious economic loss by reducing grain nutritive value and animalproduction (Wu, 2004). Among the aflatoxins, aflatoxin B1 (AFB1) isone of the highest toxic and carcinogenic compounds (Leontopou-los et al., 2003).

In order to degrade AFB1, a lot of AFB1-degrading methods suchas physical, chemical and biological treatments have been used(Galvano et al., 2001; Ali et al., 2010). However, most of these

methods are impractical or potentially unsafe, due to the formationof toxic residues or derivatives, low organoleptic qualities, andsmall-scale processing. Therefore, new practical and effectivemethods to degrade AFB1 are required. One of the most promisingmethods is to select the microbes or enzymes for degrading AFB1.Some species of microbes such as lactic acid bacteria, Bacillus sub-tilis, Aspergillus niger, Aspergillus parasiticus, Trichoderma viride,Rhodococcus erythropolis and other fungi have been reported withdifferent AFB1-degrading ability (Teniola et al., 2005; Albertset al., 2006; Cao et al., 2011). Liu et al. (1998) demonstrated thataflatoxin detoxifizyme obtained from Armillariella tabescensshowed AFB1 detoxification activities. Liang et al. (2008) reportthat the crude enzyme isolated from Stenotrophomonas could de-grade AFB1 by 85.7%. Even though there are some reports aboutAFB1 degradation by probiotics or enzymes individually in vitro,the cooperation between probiotics and AFB1-degrading enzymeand their applications in animal production have not beenreported.

In order to testify the availability of the combination, feedingand slaughter experiments of broilers were used. Through analyz-ing effects of the combination on AFB1 metabolism, production

R.-y. Zuo et al. / Food and Chemical Toxicology 59 (2013) 470–475 471

performance, antioxidant activity, hepatic microscopic structureand different hepatic enzyme gene expressions, the mechanismsfor detoxification and pathogenesis of AFB1 will be explored toeliminate the hazards of AFB1 in the future.

2. Materials and methods

2.1. Microbes and preparation

Aspergillus flavus (A. flavus), Lactobacillus casei (L. casei), Bacillus subtilis (B. sub-tilis) and Pichia anomala (P. anomala) were purchased from China General Microbi-ological Culture Collection Center (CGMCC), incubated and prepared according topublished protocols (Yin et al., 2012). AFB1 used in vitro was purchased from Sigma.Aspergillus oryzae (A. oryzae) selected from nature for producing AFB1-degrading en-zyme was identified with 26S rDNA and registered as CGMCC5817 in CGMCC. Thepotato dextrose agar (PDA) for A. flavus and A. oryzae incubation was prepared asfollows (g/L): soluble starch 6, glucose 20, yeast extract 2, peptone 5, KH2PO4 2,MgSO4 0.3 and agar 20 (Agar was removed in liquid-state medium). The mediumwas autoclaved at 121 �C for 20 min.

The addition of combined probiotics and AFB1-degrading enzyme at 0.15% levelin broiler diets was referred to the former study, in which it contained L. casei(2 � 109 colony forming unit (CFU)/g), B. subtilis (1 � 109 CFU/g), P. anomala(2 � 109 CFU/g) and AFB1-degrading enzyme (135 U/g) (Zuo, 2012). The amountof enzyme that could degrade 1 ng AFB1 per min was defined as one unit (Caoet al., 2011).

2.2. AFB1 determination and moldy corn meal preparation

RIDASCREEN� AFB1 30/15 test kit (R-Biopharm, Germany) was used to deter-mine AFB1 contents. AFB1 standard solutions used for making the calibration curvecontained 0, 1, 5, 10, 20 and 50 lg/L (ppb), and all of them were included in the en-zyme-linked immunosorbent assay (ELISA) test kit.

Eight-hundred grams corn meal passing through 2.36 mm apertures of screenwere put in a 2 L flask, added with 400 mL distilled water, and then autoclaved.The medium was inoculated with 20 mL A. flavus at 1 � 108 spores/mL, and incu-bated at 28 �C for 7 d. The incubated corn meal was autoclaved to kill A. flavus, driedand ground by passing 0.85 mm aperture of screen for animal feeding experiment.The AFB1 concentrations in moldy corn meal were estimated as 3961 lg/kg (ppb).

2.3. Animals and managements

Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into5 groups, 15 broilers for each group, 3 broilers in one cage. The basal diet was for-mulated to meet nutrient requirements according to NRC (1994). Diets and waterwere given to the birds ad libitum. The experimental period was 30 d. At the endof the feeding experiment, five chickens from each group were slaughtered for fur-ther analyses. Body weight, feed intake and diarrhea rates were recorded. The feed-ing experiment was designed as follows:

� Group A: Basal diet, determined as 46.80 lg/kg AFB1.� Group B: Basal diet added with 400 lg/kg AFB1, determined as 445.64 lg/kg

AFB1.� Group C: Basal diet added with 200 lg/kg AFB1 plus 0.15% combination of pro-

biotics and AFB1-degrading enzyme, determined as 239.72 lg/kg AFB1.� Group D: Basal diet added with 400 lg/kg AFB1 plus 0.15% combination of pro-

biotics and AFB1-degrading enzyme, determined as 441.49 lg/kg AFB1.� Group E: Basal diet added with 800 lg/kg AFB1 plus 0.15% combination of pro-

biotics and AFB1-degrading enzyme, determined as 855.54 lg/kg AFB1.

2.4. Determinations of nutrient metabolic rates

At the end of the 30 d feeding experiment, metabolic experiment was per-formed, in which feed intake was recorded daily. Fresh feces were collected withoutcontamination from each of 5 birds in each group for 3 d, 3 collecting times daily.The fecal samples of each bird from 3 d collections were dried, ground and mixed.Crude protein (CP), crude fat (CF), calcium (Ca) and phosphorus (P) in diets and ex-creta were determined with Kjeldahl, ether extract, potassium permanganate(KMnO4) and ammonium molybdate ((NH4)6Mo7O24) protocols, respectively (Jur-gens, 1997). The calculation of nutrient metabolic rates was made as follows: Nutri-ent metabolic rate = (nutrient content in diets � nutrient content in excreta)/nutrient content in diets.

2.5. Determination of antioxidant indexes and AFBl metabolism

After the feeding experiment, five 73-day-old broilers from each group weresacrificed. The serum, hepatic homogenates and chest muscle were prepared forthe assays of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), totalantioxidant capacity (T-AOC) and AFB1 concentrations. SOD activity was analyzed

by monitoring inhibition rate of reducing nitroblue tetrazolium (NBT). GSH-Pxactivity was determined according to the former method (Shi et al., 2006). T-AOCwas based on measuring the fluorescence decay of R-Phycoerythrin induced bythe peroxyl radicals obtained with thermal decomposition of azo diisobutyl ami-dine (Debora et al., 2010).

2.6. Hepatic sample preparation for microscopic analyses

The hepatic internal lobes from each group were taken, cut at 1 cm3, cleanedwith physiological saline, dried with filter paper. After formalin fixation, the sam-ples were prepared in the process of dehydration, transparence, steeping wax andembedding. They were cut at 0.6 lm thickness with microtome, put into water bath(42–45 �C), and then baked and fixed on slides. The slides were stained with hema-toxylin-eosin, treated with dimethyl benzene, and analyzed with microscope.

2.7. Gene expression analyses

The livers of 73-day-old male broilers in group A, B and D were selected todetermine gene expression. The GeneChip� Chicken Genome Array (Affymetrix,Santa Clara, CA, USA) containing 32,773 transcripts corresponding to more than28,000 chicken genes were used. The total RNA from three chicken livers in eachgroup was prepared by using TRIzol reagent (Life technologies, Carlsbad, CA,USA), further purified by RNeasy mini kit and RNase-Free DNase Set (Qiagen, Hil-den, Germany), and hybridized at 42 �C for 16 h. The reverse transcription was car-ried out by using GeneChip 3’IVT Express Kit (Affymetrix, Santa Clara, CA, USA). Themicro-array slides were scanned by GeneChip� Scanner 3000 and Command Con-sole Software 3.1 with default settings. Raw data were normalized by MAS 5.0 Algo-rithm Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, USA). Thedifferential level was represented by a log2 intensity ratio. Functional categorizationof gene expression clusters was performed by using Shanghai Bio Corporation (SBC)Gene Ontology (GO) Tools of China (http://sas.ebioservice.com).

2.8. Statistical analyses

Experimental data were expressed as means and standard errors (Means ± SE).The data were analyzed using the ANOVA procedures of Statistical Analysis Systemsinstitute (SAS 8.0). Duncan’s multiple range test was used to evaluate treatmentmeans. The results were considered statistically significance at p < 0.05.

3. Results

3.1. Effect of the combined probiotics with AFBl-degrading enzyme onbroiler production performance

Table 1 indicates that the diets added with combinations of pro-biotics and AFBl-degrading enzyme could significantly relieve thenegative effect of AFB1 on chicken’s production performance. Com-pared with group A and C, the average daily gain (ADG), averagedaily feed intake (ADFI) and feed conversion rate (FCR) were signif-icantly decreased in group B, D and E (p < 0.05). ADG (40.31 vs.39.06 g) and FCR (2.95 vs. 3.06) in group A were almost the sameas that in group C (p > 0.05), which showed that addition of0.15% combination of probiotics and AFBl-degrading enzyme inbroiler diet added with 200 lg/kg AFBl could remove AFBl hazardscompletely. There were the same ADG (�3.26 vs. �3.73 g) betweengroup B and E (p > 0.05), inferring that addition of probiotics andAFBl-degrading enzyme could increase broiler’s tolerance abilityto AFBl by 1-fold (400 vs. 800 lg/kg). In addition, ADG and FCRin group D were significantly higher than that in group B(p < 0.05), indicating that the combination could relieve AFBl haz-ards effectively.

Table 2 shows that organic matter, CP, Ca and P metabolic ratesin group B were lower than that in other groups (p < 0.05), indicat-ing that addition of probiotics and AFB1-degrading enzyme couldimprove nutrient metabolic rates of broilers. Calcium and P meta-bolic rates in group B and E were negative, signifying that medium-dose AFB1 addition without feed additive or high-dose AFB1 addi-tion with feed additive could not relieve AFB1 negative effect andcaused serious metabolic problems for Ca and P.

Table 1Effect of probiotics and AFB1-degrading enzyme on production performance of broilers.

Items Group A Group B Group C Group D Group E

Initial weight (g) 1265.33 ± 20.45A 1280.67 ± 14.01A 1299.33 ± 21.20A 1309.67 ± 10.92A 1308.00 ± 10.67AFinal weight (g) 2474.67 ± 76.78A 1183.00 ± 50.64C 2471.00 ± 38.03A 1685.33 ± 99.51B 1196.00 ± 55.84CADG (g) 40.31 ± 3.15A �3.26 ± 1.24C 39.06 ± 1.61A 12.52 ± 4.45B �3.73 ± 1.85CADFI (g) 118.97 ± 1.97A 55.78 ± 2.07C 119.48 ± 5.96A 73.88 ± 7.55B 55.70 ± 2.59CFCR 2.95 ± 0.24A �17.13 ± 9.03B 3.06 ± 0.14A 5.90 ± 1.66A �14.92 ± 20.98BDiarrhea (%) 1.11 ± 0.99A 1.56 ± 0.54A 0.89 ± 0.83A 0.67 ± 0.54A 0.89 ± 0.44A

Note: The different letters in the same rows mean significant difference (p < 0.05), while the same letters in the same rows mean insignificant difference (p > 0.05).

Table 2Effect of probiotics and AFB1-degrading enzyme on nutrient metabolic rates of broilers (%, n = 5).

Items Group A Group B Group C Group D Group E

Organic matter 66.13 ± 0.11B 57.04 ± 0.25A 62.75 ± 0.28AB 62.46 ± 0.41AB 61.92 ± 0.51ABCP 73.01 ± 0.83C 23.81 ± 1.15A 67.60 ± 1.21C 66.37 ± 1.31C 59.09 ± 1.13BCF 87.80 ± 1.02AB 85.33 ± 0.92AB 90.99 ± 0.61B 84.94 ± 0.56AB 77.58 ± 0.85ACa 23.86 ± 0.68D �11.71 ± 1.61A 24.45 ± 0.71D 6.57 ± 0.56C �7.69 ± 1.48BP 12.31 ± 2.11D �25.43 ± 3.79A 9.24 ± 0.77C 6.57 ± 0.74C �1.56 ± 0.33B

Note: The different letters in the same rows mean significant difference (p < 0.05), the same letters in the same rows mean insignificant difference (p > 0.05).

472 R.-y. Zuo et al. / Food and Chemical Toxicology 59 (2013) 470–475

3.2. Antioxidant indexes, hepatic cellular structure changes, and AFB1

contents in liver, serum and chest muscle

Table 3 shows that hepatic and serum SOD, GSH-Px and T-AOCactivities in group A, C, D and E were significantly increased exceptfor serum T-AOC activity, compared with group B (p < 0.05). Inaddition, supplementation of probictics and AFB1-degrading en-zyme in chicken diets could reduce AFB1 concentrations in liver,serum and chest muscle (p < 0.05).

Fig. 1 indicated that the eigen structure and profile of hepaticcells were clear, and hepatic cellular nuclei were big and roundin group A. A part of hepatic eigen structures disappeared, and alot of hepatic cellular necroses around with many reticulocytes,macrophagocytes and mechanocytes were found in group B andE. The degree of hepatic cellular necroses in 5 groups was: groupE > group B > group D > group C > group A. It was inferred thatthe combination of probiotics and AFB1 degrading enzyme wasable to protect liver from damage caused by AFB1.

3.3. Determination of enzyme gene expressions in broiler livers

The results showed that many enzyme gene expressions in broi-ler livers were changed due to AFBl addition as well as the

Table 3Antioxidant indexes and AFB1 metabolism of broilers (n = 3).

Items Group A Group B

In serumSOD (U/mL) 68.08 ± 3.49C 61.09 ± 5.57CGSH-Px (U/mL) 555.12 ± 58.17A 422.90 ± 52.20BT-AOC (U/mL) 16.12 ± 2.00A 15.01 ± 0.94AAFB1 contents (lg/L) 5.36 ± 0.74A 8.68 ± 1.64B

In liverSOD (U/g protein) 79.65 ± 0.09B 60.41 ± 0.71CGSH-Px (U/g protein) 17.60 ± 1.89A 12.75 ± 1.22BT-AOC (U/g protein) 21.70 ± 2.51A 10.62 ± 0.14BAFB1 contents (lg/kg) 5.82 ± 0.02B 7.02 ± 0.80C

In chest muscleAFB1 contents (lg/kg) 0.86 ± 0.34A 1.63 ± 0.49B

Note: The different letters in the same rows mean significant difference (p < 0.05), the s

combined probiotics with AFBl-degrading enzyme addition. Com-pared with group B, the number of up-regulated and down-regu-lated genes in group A was 17,383 and 21,056 over 1-foldregulation, 850 and 1126 over 2-fold regulations, 366 and 409 over3-fold regulations, respectively. Compared with group D, the num-ber of up-regulated and down-regulated genes in group A was18,319 and 20,120 over 1-fold regulation, 1398 and 1028 over 2-fold regulations, 627 and 394 over 3-fold regulations, respectively.Compared with group D, the number of up-regulated and down-regulated genes in group B was 20,184 and 18,255 over 1-fold reg-ulation, 1723 and 961 over 2-fold regulations, 1036 and 386 over3-fold regulations, respectively.

Because there were so many candidate genes, only the up-reg-ulated and down-regulated genes over 3 folds were studied. Thegenes related with oxidoreductase, apoptosis, cell growth, immunesystem and metabolic process were considered especially in thisstudy.

Using microarray analysis, we found 447 genes involved in oxi-doreductase family. The number of up-regulated and down-regu-lated genes over 3 folds in A/B, A/D and B/D was 19, 26 and 26,respectively. Table 4 indicates that there were 5 genes up-regu-lated and 3 genes down-regulated over 3 folds in group A and D,compared with that in group B.

Group C Group D Group E

115.79 ± 3.40A 101.55 ± 7.85B 88.78 ± 8.20B521.40 ± 37.93A 484.37 ± 34.04A 444.87 ± 29.11AB

13.53 ± 2.38A 14.76 ± 1.72A 12.66 ± 0.71A5.28 ± 0.30A 5.58 ± 1.04A 8.81 ± 0.24B

87.37 ± 13.36B 80.58 ± 9.24B 96.04 ± 19.51A16.75 ± 2.30A 16.47 ± 2.01A 15.49 ± 0.33A22.38 ± 5.22A 22.38 ± 0.59A 22.55 ± 6.05A

4.01 ± 0.31A 5.62 ± 0.81B 5.39 ± 1.01B

0.97 ± 0.28A 1.14 ± 0.11A 1.41 ± 0.15B

ame letters in the same rows mean insignificant difference (p > 0.05).

Group A Group B Group C

Group D Group E

Fig. 1. Photomicrographs of broiler hepatic cells (�400).

Table 4Effect of probiotics and AFB1-degrading enzyme on gene expression quantity ofoxidoreductase in broiler liver.

ID Log2 ratio

A/B A/D B/D

Gga.1898.1.S1 5.3774 0.2934Gga.4401.1.S1-a 5.0859 10.1975GgaAffx.21824.1.S1 0.0794 0.0665GgaAffax.21843.2.S1 6.9678 16.7570Gga.12729.1.S1 0.2807 3.9970GgaAffx.12957.1.S1 7.8133 0.1126GgaAffx.8040.1.S1-s 0.2236 0.0829Gga.9528.1.S1 0.1012 13.4037GgaAffx.10453.1.S1-s 10.7902 7.3152GgaAffx.12133.1.S1 11.7467 9.0066Gga.10928.1.S1 48.7539 43.1877GgaAffx.12935.1.S1-s 6.8757 5.3880Gga.10231.1.S1 0.2702 0.2977Gga.11710.1.S1 293.1239 0.0035Gga.7259.1.A1 52.9196 0.0817Gga.8152.1.S1 0.2926 4.9456Gga.1290.1.S1 4.6817 0.2009GgaAffx.6820.1.S1 24.8639 6.6983

Note: When the data of A/B, A/D and B/D are more than 1.0000, they mean the genesin group A, A and B are up-regulated over 3 folds; otherwise, they are down-reg-ulated over 3-fold. Only the data with both changes (A/B and A/D, A/B and B/D, or A/D and B/D) are listed in the table.

Table 5Effect of probiotics and AFB1-degrading enzyme on gene expression quantity for cellgrowth, cell death and immune system process in broiler liver.

ID Log2 ratio

A/B A/D B/D

GO: 0016049 cell growthGga.9509.1.S1 3.5951 4.8032Gga.811.1.S1 8.6996 6.3521Gga.5710.2.S1-a 4.1364 5.2039

GO: 0008219 cell deathGga.961.2.S1 4.3787 0.2359Gga.8409.1.S1 4.1680 0.1094Gga.6713.1.S1 3.5223 4.6477Gga.9334.2.S1-a 4.7802 0.3173Gga.3545.1.S2 28.1216 17.1773Gga.10350.1.S1 3.8346 3.1734Gga.713.1.S1 12.8682 13.5820Gga.16038.1.S1 0.2545 3.6569Gga.3903.1.S1 14.5511 21.7538GgaAffx.12451.1.A1-s 0.2820 7.1506GgaAffx.12935.1.S1-s 6.8757 5.3880GgaAffx.13009.1.S1-a 3.0834 3.6384

GO: 0002682 regulation of immune system processGga.16038.1.S1 0.2545 3.6569Gga.10155.1.S1 4.1110 0.3130GgaAffx.7591.1.S1 12.5507 0.0485GgaAffax.12451.1.S1-s 0.2820 7.1516

Note: When the data of A/B, A/D and B/D are more than 1.0000, they mean the genesin group A, A and B are up-regulated over 3 folds; otherwise, they are down-reg-ulated over 3-fold. Only the data with both changes (A/B and A/D, A/B and B/D, or A/D and B/D) are listed in the table.

R.-y. Zuo et al. / Food and Chemical Toxicology 59 (2013) 470–475 473

There were 44 enzyme genes involved in the family of cellgrowth. The number of up-regulated and down-regulated genesover 3 folds in A/B, A/D and B/D was 2, 4 and 6, respectively. Table 5indicates that there were 3 genes down-regulated over 3 folds ingroup D, compared with that in group A and B.

There were 271 enzyme genes involved in the family of celldeath. The number of up-regulated and down-regulated genes over3 folds in A/B, A/D and B/D was 6, 11 and 20, respectively. It wasindicated that there were 9 genes down-regulated and 3 genesup-regulated over 3 folds in group D, compared with that in groupB (Table 5).

There were 94 enzyme genes involved in the family of immunesystem. The number of up-regulated and down-regulated genesover 3 folds in A/B, A/D and B/D was 6, 1 and 11, respectively. There

were 2 genes down-regulated and 2 genes up-regulated over 3folds in group A and D, compared with that in group B (Table 5).

There were 1082 enzyme genes involved in the family of meta-bolic process. The number of up-regulated and down-regulatedgenes over 3 folds in A/B, A/D and B/D was 30, 37 and 54, respec-tively. Table 6 shows that there were 9 genes up-regulated and 4genes down-regulated over 3 folds in group A and D, comparedwith that in group B.

Table 6Effect of probiotics and AFB1-degrading enzyme on gene expression quantity ofmetabolic process in broiler liver.

ID Log2 ratio

A/B A/D B/D

Gga.137.1.S1 4.1719 5.6625Gga.961.2.S1 4.3787 0.2359Gga.4939.1.S1-s 0.0945 0.3156 3.3409Gga.6220.1.S1-a 5.5765 0.2354GgaAffx.21787.1.S1-s 0.3067 3.1248 10.1891GgaAffx.12957.1.S1 7.8133 0.1126Gga.9509.1.S1 3.5951 4.8032Gga.10350.1.S1-s 3.8346 3.1734GgaAffx.21783.1.S1-s 5.1228 3.4753Gga.3393.2.S1.a 6.3540 11.6719Gga.1313.1.S1-s 3.9730 7.0126Gga.701.1.S1-s 3.9713 6.4415Gga.755.1.S1 3.1968 0.2705Gga.4131.1.S1 0.2914 0.1101Gga.16038.1.S1 0.2545 3.6569Gga.3903.1.S1 14.5511 21.7538Gga.15899.1.S1 7.6050 5.7876GgaAffx.7591.1.S1 12.5507 0.0485GgaAffx.12451.1.S1-s 0.282 7.1516Gga.18259.1.S1-s 0.1545 0.0371Gga.19230.1.S1-s 3.0252 0.2753GgaAffx.7682.1.S1 3.5440 0.3220GgaAffx.11214.1.S1 3.4940 6.9464Gga.4012.1.S1 14.3447 5.9802Gga.10155.1.S1 4.1110 0.3130GgaAffx.129571.1.S1 7.8133 0.1126

Note: When the data of A/B, A/D and B/D are more than 1.0000, they mean the genesin group A, A and B are up-regulated over 3 folds; otherwise, they are down-reg-ulated over 3-fold. Only the data with both changes (A/B and A/D, A/B and B/D, or A/D and B/D) are listed in the table.

474 R.-y. Zuo et al. / Food and Chemical Toxicology 59 (2013) 470–475

4. Discussion

AFB1 is one of the most toxic mycotoxin in the world, with neg-ative effect on animal productions and economic profits (Boldenand Jensen, 1985; Swamy and Devegowda, 1998; Sumit and Roger,2010). It was reported that the broiler diets containing 100 lg/kgof AFB1 had significant effect on ADG and FCR of broilers (Anongand Suparat, 2006; Shi et al., 2006). Duo to universal AFB1 contam-ination in feedstuffs, chemoprevention strategies aimed at reduc-ing AFB1 toxicity in animal diets have become the subject ofnumerous studies (Klein et al., 2003; Guarisco et al., 2008). Severalchemopreventions have been evaluated in poultry for reducingsymptoms of aflatoxicosis. Cao et al. (2011) reported that supple-mentation of aflatoxin detoxifizyme in 100 lg/kg AFB1 diet im-proved ADFI and ADG of chickens. This result indicated thataddition of probiotics and AFB1-degrading enzyme in chicken dietwith less than 240 lg/kg of AFB1 had the ability to detoxify AFB1

and relieve its negative effect on chicken’s production perfor-mance. When AFB1 concentration in broiler diet was more than856 lg/kg, the additives did not work well for degrading AFB1. Thisdisagreed with the report of Tejada-Castaneda et al. (2008), inwhich ADG and FCR of broilers fed with AFB1 (800 lg/kg) in thediet were significantly improved by Nocardia corynebacteroide. Itmay be due to the high AFB1 concentrations exceeding the detox-ificating ability of the 0.15% combined probiotics and AFB1-degrad-ing enzyme in this study.

The liver is the main target organ for AFB1 metabolism. Chronicexposure to low levels of AFB1 in foodstuffs will induce oxidativestress both in vivo and in vitro, even cause liver fibrosis and cancer(Souza et al., 1999). Biological degradation of AFB1 was consideredto be the most promising method, which can degrade AFB1 to non-toxic or low toxic metabolites through microbial actions (Line andBrackett, 1995) or AFB1-degrading enzymes (Liu et al., 1998;

Alberts et al., 2006). This study has testified effect of the feed addi-tive on protecting liver from damage induced by AFB1.

Effect of AFB1 on antioxidant enzyme activity is one of the majorproblems for animal health. Antioxidant capacity has direct effecton animal health and anti-tumor ability. SOD and GSH-Px are themain antioxidant enzymes in animal body, which scavenge the un-wanted O2�, H2O2 and ROOH produced by free radicals. SOD cancatalyze superoxide radical dismutation, and GSH-Px can decom-pose hydrogen peroxide (Wills, 1966). The decreased activity ofthese enzymes can induce high contents of free radicals, resultingin cell damage. The damaged hepatic cell and low enzyme activitycaused by AFB1 in this study proved the above inference. Shyamalet al. (2010) reported that the potent antioxidant properties wouldhelp to reduce cell damage caused by AFB1, which is related to thisstudy. Shi et al. (2006) reported that the diet containing 100 lg/kgAFB1 significantly decreased activities of the liver SOD and GSH-Px.In this study, SOD, GSH-Px and T-AOC activities in liver were de-creased, and hepatic cells were damaged in chicks fed with AFB1

without additives, while addition of the combined probiotics andAFBl-degrading enzyme had the ability to keep liver’s antioxidantcapacity at the regular levels, and to keep liver from AFB1 damageto some extents.

The samples collected from birds showed detectable levels ofAFB1 in liver, serum and muscles (Bintvihok and Davitiyananda,2002). This study indicated that addition of probictics and AFB1-degrading enzyme in chicken diets could reduce AFB1 concentra-tions in liver, serum and chest muscle, in agreement with the for-mer reports (Zahid et al., 2010; Hussain et al., 2010). AFB1 residuesin poultry tissues may pose a risk to consumer health, so AFB1 bio-degradation must receive special attention when its concentrationin feedstuffs is higher than the limited amounts.

Acute or chronic aflatoxicosis in birds will result in decreasedfeed efficiency, immunosuppressant, and hepatotoxicosis (Vermaet al., 2004). AFB1 can cause cell damages by affecting antioxidantenzyme activity, liver function, immune system, metabolic processand cytochrome P450s (CYP1A1) gene. At present, the pathoge-netic mechanism of AFB1 was unclear. The extreme sensitivity ofpoultry to AFB1 is associated with efficient hepatic cytochromeP450-mediated bioactivation (Yarru et al., 2009a; Sumit and Roger,2010). Cytochrome P450s is a group of oxidases that catalyzes thebiotransformation of a wide variety of xenobiotics. Liver P450s isespecially efficient toward AFB1 bioactivation compared to othertissues (Sumit and Roger, 2010). Therefore, study on gene expres-sions in broiler liver affected by AFB1 and addition of the combinedprobiotics and AFBl-degrading enzyme has become more and moreimportant for revealing the pathogenetic mechanism of AFB1 andeliminating its hazard.

It was reported that hepatic gene expression of antioxidant en-zymes such as SOD, GSH-Px, GST and cytochrome P450 weredown-regulated or affected in chicks fed with diets containingAFB1 (Gowda et al., 2008; Yarru et al., 2009b). In this study, 5 genesfor antioxidant enzyme activity in group A and D were up-regu-lated compared with group B, which may be responsible for thehigher concentrations of SOD, GSH-Px and T-AOC in broiler liver.In addition, all the genes for cell growth were down-regulated bythe feed additive, but ADG in group D was the same as in groupA. The reason may be due to the hysteretic nature of gene tran-scription (Towle, 1995), which needs further study.

Most genes for regulating metabolic process in group B weredown-regulated, compared with group A and D, indicating thataddition of the combined probiotics and AFBl-degrading enzymecould relieve the negative impact of AFBl and improve nutrientmetabolic rates. They correlate with the results of metabolic exper-iment in this study, but disagree with the previous report, in whichsome of the genes associated with carbohydrate metabolism andprotein metabolism were up-regulated in birds fed 2 mg/kg of

R.-y. Zuo et al. / Food and Chemical Toxicology 59 (2013) 470–475 475

AFB1 in order to compensate for the reduced caloric intake and en-ergy utilization (Yarru et al., 2009b). It is probable that the differ-ent genes related with nutrient metabolic process have thedifferent responses induced by AFB1 hazards.

Most genes for cell death were down-regulated by the feedadditive, maybe due to the fact that adding probiotics and AFB1-degrading enzyme in chicken diet could relieve the hepatic lesionsand protect cells from damages by AFB1, which was proven by thehepatic cell photomicrographs, and consistent with an earlier re-port (Voss et al., 2001). The previous report showed that AFB1 atdietary concentrations of 100 lg/kg or more would cause a signif-icant reduction in immune response (Verma et al., 2004). For im-mune family in this study, the up-regulated and down-regulatedgenes are correspondent in group A and D, meaning that the feedadditive is able to remove AFB1 hazards and regulate immune geneexpressions at the same levels.

It is very interested to find that all the up-regulated or down-regulated genes in group A and D had the same changing tendencywhen compared with group B, indicating that addition of probio-tics and AFB1-degrading enzyme in broiler diets could regulategene expressions to the normal levels. It is concluded that the com-bination of probiotics and AFB1-degrading enzyme is able to de-grade AFB1, relieve its negative effect on nutrient availability,hepatic cell structure, antioxidant activity, and up-regulate ordown-regulate hepatic enzyme gene expressions for keeping thenormal production performances of broilers.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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