af+microarray

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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

2009 Poultry Science 88:360371doi:10.3382/ps.2008-00258

Key words:aflatoxin B1, gene expression, broiler, liver, microarray

ABSTRACT The objective of this study was to deter-mine the effects of dietary aflatoxin B1(AFB1) on he-patic gene expression in male broiler chicks. Seventy-five1-d-old male broiler chicks were assigned to 3 dietarytreatments (5 replicates of 5 chicks each) from hatch tod 21. The diets contained 0, 1 and 2 mg of AFB1/kg offeed. Aflatoxin B1reduced (P< 0.05) feed intake, BWgain, serum total proteins, and serum Ca and P, butincreased (P< 0.01) liver weights in a dose-dependentmanner. Microarray analysis was used to identify shifts

in genetic expression associated with the affected physi-ological processes in chicks fed 0 and 2 mg of AFB 1/kg of feed to identify potential targets for pharmaco-logical/toxicological intervention. A loop design wasused for microarray experiments with 3 technical and4 biological replicates per treatment group. Ribonucleicacid was extracted from liver tissue, and its qualitywas determined using gel electrophoresis and spectro-photometry. High-quality RNA was purified from DNA

contamination, reverse transcribed, and hybridized toan oligonucleotide microarray chip. Microarray datawere analyzed using a 2-step ANOVA model and vali-dated by quantitative real-time PCR of selected genes.Genes with false discovery rates less than 13% and foldchange greater than 1.4 were considered differentiallyexpressed. Compared with controls (0 mg of AFB1/kg),various genes associated with energy production andfatty acid metabolism (carnitine palmitoyl transferase),growth and development (insulin-like growth factor 1),

antioxidant protection (glutathione S transferase), de-toxification (epoxide hydrolase), coagulation (coagula-tion factors IX and X), and immune protection (inter-leukins) were downregulated, whereas genes associatedwith cell proliferation (ornithine decarboxylase) wereupregulated in birds fed 2 mg of AFB1/kg. This studydemonstrates that AFB1exposure at a concentrationof 2 mg/kg results in physiological responses associatedwith altered gene expression in chick livers.

Toxicological and gene expression analysis of the impact of aflatoxin B1on hepatic function of male broiler chicks

L. P. Yarru,* R. S. Settivari,* E. Antoniou,* D. R. Ledoux,*1and G. E. Rottinghaus

*Department of Animal Sciences, and Veterinary Medical Diagnostic Laboratory,University of Missouri, Columbia 65211

INTRODUCTION

Mycotoxins are naturally occurring toxic secondarymetabolites of fungi that may be present in food in-gredients (Kuiper-Goodman, 1995). Several mycotoxinshave been associated with various animal and poultrydiseases. Mycotoxins encompass a wide spectrum of dif-ferent chemical structures and affect many target or-gans such as liver and kidney and systems such as thenervous and immune systems (Kuiper-Goodman, 1995).A major contaminant of common feed ingredients used

in poultry rations is the aflatoxins (AF; Smith et al.,1995). Aflatoxins are a class of mycotoxins produced bythe fungi Aspergillus parasiticusand Aspergillus flavus(Smith et al., 1995). Aflatoxin B1(AFB1) is the most

biologically active form and causes poor performance,liver lesions, and immunosuppression in poultry (Kube-na et al., 1990; Ledoux et al., 1999). Aflatoxin B1alsoincreases free radical production, leading to oxidativedamage and lipid peroxidation, which might ultimatelylead to cell damage and death (Surai, 2002). Eraslan etal. (2005) studied the effects of AF on oxidative stressand observed a reduction in antioxidant activity inthe erythrocytes of chicks fed AF compared with con-trols. Although problems associated with AF have beenknown for decades and a great deal of research has been

conducted on the effects of AF at the animal level, verylittle research has been done at the gene level. Microar-rays are being used for global gene expression profilingto identify candidate genes and to map growth, meta-bolic, and regulatory pathways that control importantproduction traits. To date, no study has been reportedregarding the measurement of gene expression in chicksfed AFB1using microarrays. The current project willallow us to determine genes that are specifically ex-

Received June 25, 2008.Accepted September 29, 2008.1Corresponding author: [email protected]

2009 Poultry Science Association Inc.

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pressed in response to AF and thus enable us to identi-fy various pathways that control growth, development,coagulation, immune, metabolism, detoxification, andantioxidant status of broiler chicken.

MATERIALS AND METHODS

Experimental Design and Birds

Seventy-five 1-d-old (Cobb Cobb) male broilerchicks were purchased from a commercial hatchery(Hoovers Hatchery Inc., Rudd, IA), weighed, wing-banded, and assigned to pens in stainless-steel chickbatteries for 21 d. Chicks were maintained on a 24-hcontinuous light schedule and allowed ad libitum accessto feed and water. The animal care and use protocolwas reviewed and approved by the University of Mis-souri-Columbia Animal Care and Use Committee. Thechicks were randomly assigned to 3 treatments with 5replicates of 5 birds each. Mortality was recorded, andbirds were inspected daily for any health problems.

Diets

The basal diet was a commercial type corn-soybeanmeal diet formulated to meet the nutritional require-ment of growing chicks as recommended by the Na-tional Research Council (1994). Dietary treatments in-cluded A) a basal diet containing 0 mg of AFB1/kg ofdiet (control); B) a basal diet with 1.0 mg of AFB1/kgof diet; and C) a basal diet with 2.0 mg of AFB1/kg ofdiet. Aflatoxin B1was supplied by Aspergillus parasiti-cus(NRRL 2999) culture material containing 760 mg/kg of AFB1.

Sample Collection

On d 21, all birds were weighed and feed intake wasmeasured for each pen. Average feed intake and BWgain were determined. Fifteen chicks (5 replicates of3 chicks each) from each treatment were killed withcarbon dioxide, and blood was collected via cardiacpuncture for serum chemistry analysis. Liver weight ofeach bird was recorded, and a piece of liver tissue wascollected, snap-frozen in liquid nitrogen, and stored at80C for microarray and real time-PCR analyses.

Serum Biochemistry

Collected blood was centrifuged to obtain serum,which was immediately frozen until submitted for se-rum biochemistry analysis. Serum analyses includeddeterminations of serum total protein (TP), Ca, andP using standard procedures. These measurements arecomponents of a larger biochemical profile determinedat the Veterinary Medical Diagnostic Laboratory usingan auto-analyzer (Kodak Ektachem Analyzer, EastmanKodak Co., Rochester, NY).

Statistical Analysis of PhysiologicalResponses

Data were analyzed using the General Linear Modelprocedures of SAS (SAS Institute, 1996). The meansfor treatments showing significant differences in theANOVA were compared using Fishers protected leastsignificant difference procedure at a significance based

on the 0.05 level of probability.

Microarray Slide Preparation

Microarray slides containing 21,120 oligonucleotides(70 bp; representing entire chicken genome) were pur-chased from the Genomic Research Laboratory (Uni-versity of Arizona, Tucson). Before hybridization, themicroarray slides were rehydrated to increase the spotsize and to spread the DNA more uniformly withineach spot. Following rehydration for 30 s at 55C, theslides were snap-dried on a 100C hot plate surface for5 s. The slides were cross-linked using an ultraviolet

cross-linker (600 mJ). Microarray slides were incubatedin 0.2% I-block (Applied Biosystems, Foster City, CA)at 42C to decrease background and increase specificity.The slides were washed once with 0.2% SDS and twicewith water (3 min each wash). The slides were dried bycentrifugation and stored in the dark until used.

RNA Extraction

Ribonucleic acid was extracted from the liver sam-ples of birds fed 0 and 2 mg of AFB1/kg using anRNeasy Midi Kit (Qiagen Inc., Valencia, CA), puri-fied using DNase-1 (Ambion Inc., Austin, TX) and

phenol:chloroform:isoamyl alcohol (25:24:1), and con-centrated using Microcon YM30 filters (Millipore Corp.,Bedford, MA) as described previously (Settivari et al.,2006). The quality and integrity of the purified RNAwas checked through agarose gel electrophoresis, andthe quantity was measured using an ND-1000 spectro-photometer (Nanodrop Technologies, Wilmington, DE;Flanagan, 2005). The purified RNA samples were pre-served at 80C until used.

Microarray Hybridization

A loop design was used for microarray experiments,with 4 biological replicates (4 animals/treatmentgroup) and 3 technical replicates (each RNA samplerepeated on 3 different arrays; Figure 1). Concentratedand purified total RNA (15 g) from each chick liverwas reverse-transcribed to cDNA using oligo dT (5g/L; IDT DNA, Coralville, IA), random hexamers(5 g/L; IDT DNA), 25 amino-allyl dUTP/dNTP(Sigma Chemicals, St. Louis, MO), and reverse tran-scriptase (StrataScript RT, Stratagene, La Jolla, CA).The resulting cDNA was purified using Microcon-30filters (Millipore) and conjugated separately with Cy3

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ment groups represented various important pathwayssuch as detoxification, fatty acid metabolism, oxidativephosphorylation, energy production, cell proliferation,coagulation, metabolism, growth and development, im-mune response, and antioxidant activities (Tables 2 to8).

Real-Time PCR Results

Quantitative real-time PCR was used to confirm thevalidity of the microarray results. All 4 of the randomlyselected differentially expressed genes (ornithine decar-boxylase, insulin-like growth factor 1, F9, GST) hada similar expression pattern as observed in microarrayresults, thereby validating the microarray results (Fig-ure 4).

DISCUSSION

Physiological Responses to Aflatoxicosis

Decreased feed intake and BW gain along with in-creased liver weights in birds fed AFB1are consistentwith earlier reports (Huff et al., 1988; Ortatatli andO!uz, 2001; Verma et al., 2004) on the effects of AF

in young broiler chicks. Similarly, reduced levels of TP,Ca, and P in birds fed AFB1are in agreement with ear-

lier reports (Huff et al., 1988). A decrease in TP levelswould lead to decreased efficiency of the immune systembecause the key mechanisms of some immune responsesare the production of factors that kill pathogens, suchas antimicrobial peptides and proteins (Bchau andGallo, 2007). Decreased serum Ca and P levels couldprobably be one of the reasons for the leg and boneabnormalities associated with rubbery leg syndrome inbirds fed AF (Washburn et al., 1976).

Genomic Responses to AFB1

Intake of the AFB1diet was hypothesized to resultin changes in liver gene expression representing charac-teristic pathophysiology associated with aflatoxicosis.Microarray results demonstrated that the expressionof genes coding for specific physiological pathways, in-cluding detoxification, fatty acid metabolism, oxidativephosphorylation, energy production, cell proliferation,immune response, metabolism, growth and develop-ment, coagulation, and antioxidant activities were al-tered in chicks fed 2 mg of AFB1/kg of diet. The al-tered genomic responses observed in the present studycould be due to the direct effects of stress associatedwith AF. These genomic effects and their relationship

Figure 2.Effect of aflatoxin B1(AFB1) on feed intake, BW gain,and relative liver weights of broiler chicks. acBars with no commonletter are significantly different (P< 0.05).

Figure 3.Effect of aflatoxin B1(AFB1) on serum P, Ca, and totalprotein. acBars with no common letter are significantly different (P< 0.05).

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to the observed physiological responses are expected toprovide additional insight into the pathophysiology ofaflatoxicosis.

Oxidative Phosphorylation and Energy Produc-tion. Genes associated with oxidative phosphorylationand adenosine triphosphate (ATP) synthesis weredownregulated in liver of chicks fed 2 mg of AFB1/kg ofdiet (Table 2). Genes such as adrenodoxin mitochondrialprecursor (adrenal ferredoxin), cytochrome P450 2C5,cytochrome P450 2P3, and nicotinamide adenine dinu-cleotide phosphate (NADH)-cytochrome b5 reductaseplay a major role in electron transport. Downregulationof these genes in chicks fed 2 mg of AFB1/kg of dietcould result in impaired oxidative phosphorylation as

well as electron leak, resulting in formation of superox-ide radicals. The major purpose of electron transport isATP synthesis, and therefore we speculate that oxida-tive phosphorylation and ATP synthesis pathways aremore affected in chicks fed 2 mg of AFB1/kg leadingto decreased energy production. Carnitine palmitoyltransferase 1A transports long-chain fatty acids intomitochondria for energy release, especially when hepat-ic glycogen is depleted during starvation (Shin et al.,2006). Long-chain fatty acids cross the outer and innermembranes of mitochondria with the help of carnitinepalmitoyl transferase and undergo -oxidation. The

resulting end product enters the Krebss cycle in themitochondrial matrix. The by-products of the Krebsscycle, NADH, and flavin adenine dinucleotide enter theelectron transport chain, resulting in ATP synthesis.Because the carnitine palmitoyl transferase gene was

Table 2.Differentially expressed genes associated with oxidative phosphorylation and energy synthesis in B 1-fed chicks comparedwith control birds at the end of 21-d treatment period1

Transcript IDGenesymbol Gene name Gene ontology

Ratio,aflatoxin/control(log values)

FDR,%

ENSGALT00000000598.1 KCNA10 Shaker subfamily potassiumchannel

Voltage-gated ion channelactivity

Up, 0.63 12.76

ENSGALT00000027702.1 FDX1 Adrenodoxin mitochondrial

precursor (adrenal ferredoxin)

Electron transport Down, 0.49 12.66

ChEST492f11 CYP2C45 Cytochrome p-450 2c45 Electron transport Down, 1.59 12.66ChEST371g7 CYP2P3 Cytochrome P450 2P3 Electron transport Down, 0.77 12.93ChEST1027e3 ACVR1 Collagen alpha1 Anion transport, ion transport Up, 0.69 12.57ENSGALT00000011328.1 UBE2I BMP/retinoic acid-inducible

neural-specific proteinATP binding, pyrophosphataseactivity

Up, 1.43 12.59

ENSGALT00000024891.1 ABCA1 ATP-bindingcassettesub-familyA

ATP-binding cassettetransporter

Up, 0.93 12.64

ENSGALT00000008501.1 CYB5R NADH-cytochromeb5 reductase


CK607798 CPT Carnitine palmitoyl transferase Transport long-chain fattyacids into mitochondria

Down,0.57 12.56

NM_008898 Por P450 (cytochrome)

oxidoreductase

Electron transport Up, 0.20 12.50

ENSGALT00000020816.1 IGF1 Insulin-like growth factor Iprecursor (IGF-I; Somatomedin)

Growth-promoting activity Down, 1.63 12.55

ENSGALT00000024891.1 ABCA1 ATP-bindingcassettesub-familyA

ATP-binding cassettetransporter

Up, 0.93 12.64

ENSGALT00000013008.1 GRB2 Growth factor receptor-boundprotein 2

Growth factor stimulation Down, 0.46 13.00

1FDR = false discovery rate; NADH = nicotinamide adenine dinucleotide phosphate; ATP = adenosine triphosphate; BMP = bone morphogenicprotein.

Figure 4.Validation of micro array results with real-time PCR.Treatment and control birds consisted of 3 biological replicates. IGF-1= insulin-like growth factor 1; OD = ornithine decarboxylase; F9 =coagulation factor IX; GST = glutathione S-transferase; RT-PCR =real-time-PCR.



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downregulated in chicks fed AFB1, a decreased numberof long chain fatty acids would be able to cross themitochondrial membranes leading to a decrease in ATPproduction.

Metabolism and Growth. Differentially expressedgenes associated with metabolism are listed in Table3. To compensate for the reduced caloric intake andenergy utilization, some of the genes associated with

carbohydrate metabolism [glycogen synthase 1, lac-tate dehydrogenase H subunit, pyruvate dehydrogenasecomplex E1 component, malic enzyme 1 nicotinamideadenine diculeotide phosphate (+)-dependent cytosolic]and protein metabolism (muller glia protein tyrosinephosphatase 2, LIM domain kinase 2, presenilin 1, he-patocyte growth factor-like/macrophage stimulatingprotein, hect domain, and regulator of chromosomecondensation 1 domain 4) were upregulated in birds

fed 2 mg of AFB1/kg, likely resulting in an upregu-lation of gluconeogenic pathways. The genes involvedin fatty acid metabolism (lipoprotein lipase precursor,peroxisomal bifunctional enzyme) were downregulatedin birds fed AFB1compared with controls. As a result,fatty acids were not metabolized and they accumulatedin the liver. This could be the reason for the fatty liv-ers and increased liver weights observed in the pres-

ent study. Furthermore, downregulation of carnitinepalmitoyl transferase gene in birds fed 2 mg of AFB1/kg could contribute to fatty liver condition becauseof impaired long chain fatty acid transport into mito-chondria. Similar increases in the relative liver weightswere observed in chicks fed AF by Ortatatli and O!uz(2001) and Huff et al. (1988). Previous reports sug-gest that AF at dietary concentrations of 1 mg/kg ormore causes severe reduction in growth of broiler chicks

Table 3.Differentially expressed genes associated with metabolism in aflatoxin B1-fed chicks compared with control birds at the endof 21-d treatment period1

Transcript ID Gene symbol Gene name Gene ontology


FDR,%

ENSGALT00000021664.1 GYS1 Glycogen synthase1 Carbohydrate metabolism Up, 2.33 12.53ENSGALT00000008794.1 NAT1 N-acetyltransferase 1 (arylamine

n-acetyltransferase)Coenzyme metabolism, cofactorbiosynthesis

Up, 1.49 12.51

ENSGALT00000005829.1 GLUL Glutamine synthetase (glutamateammonia ligase)

Amine metabolism Up, 1.26 13.03

ChEST771a4 RPL5 GTP cyclohydrolase I feedbackregulator

Carboxylic acid metabolism Up, 0.76 12.57

ENSGALT00000022490.1 MULLER Muller glia protein tyrosinephosphatase 2

Cellular macromolecule metabolism Up, 1.4 12.87

ChEST1010o8 Bbox1 -butyrobetaine,2-oxoglutarate diox Amino acid derivative metabolism Up, 1.09 12.51ChEST214h19 Slc45a2 Solute carrier family 45, member 2 Lipid metabolism, steroid

metabolismUp, 0.85 12.71

ENSGALT00000017326.1 SCP-2 Nonspecific lipid-transfer protein Nucleic acid metabolism Up, 0.55 13.04ENSGALT00000007741.1 YBX1 y box binding protein 1 Nucleic acid metabolism Up, 0.61 12.74ENSGALT00000011263.1 LIMK2 LIM domain kinase 2 Protein metabolism Up, 0.54 12.52ENSGALT00000022490.1 PTPRG MULLER glia protein tyrosine

phosphatase 2Protein metabolism Up, 1.40 12.87

ChEST748i8 LDH-B Lactate dehydrogenase H subunit Carbohydrate metabolism Up, 1.58 12.08ChEST532d8 PSEN1 Presenilin 1 Protein metabolism Up, 0.69 12.72NM_001006164.1 Pemt Phosphatidylethanolamine

N-methyltransferase isoform 1Cellular lipid metabolism, lipidbiosynthesis

Up, 1.02 12.59

ENSGALT00000024882.1 lplp Lipoprotein lipase precursor Lipid metabolism Down, 2.73 12.48ChEST496l3 PDK3 Pyruvate dehydrogenase complex,

E1 componentGlucose metabolism Up, 0.78 12.53

ENSGALT00000025553.1 ME1 Malic enzyme 1 NADP(+)-dependent cytosolic

Carboxylic acid metabolism Up, 1.72 12.56

ChEST859c9 PBE Peroxisomal bifunctional enzyme Cellular lipid metabolism Down, 1.03 12.66ENSGALG0000002722.1 ALG6 Hepatocyte growth factor-like Protein metabolism Up, 0.92 12.61NM_001012591.1 HERC4 hect domain and RLD 4 Protein metabolism Up, 0.81 12.79

ENSGALG00000001139.1 PTK7 Fibroblast growth factor receptorCPE-FGFR Cellular macromoleculemetabolism, phosphorusmetabolism

Up, 0.71 12.61

ENSGALT00000001542.1 RPL35 Ribosomal protein 135 Protein metabolism Down, 0.47 12.69ENSGALT00000001334.1 RCJMB04_9m1 Lysyl-trna synthetase Amino acid metabolism Down, 0.62 12.55ChEST993m11 RPL5 Ribosomal protein 15 Cellular protein metabolism, Down, 0.49 12.61ChEST568g13 RPL7A Ribosomal protein 17a Cellular protein metabolism Down, 0.69 12.74ENSGALT00000000960.1 LOC395465 cbp/p300-interacting transactivator Nucleobase, nucleoside, nucleotide

and nucleic acid metabolismDown, 0.74 12.72

ChEST337c15 LOC395492 Aldolase a Carbohydrate catabolism Down, 1.91 12.55ENSGALT00000013370.1 RPL30 Ribosomal protein 130 Protein metabolism Down, 0.44 12.57ENSGALT00000000422.1 PKNOX2 pbx/knotted 1 homeobox 2 Nucleobase, nucleoside, nucleotide

and nucleic acid metabolismDown, 0.55 13.04

ENSGALT00000006295.1 ALAS1 5-aminolevulinate synthase Coenzyme metabolism porphyrinmetabolism

Down, 0.87 13.01

1FDR = false discovery rate; GTP = guanosine triphosphate.

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(Verma et al., 2004). Insulin like growth factor-1, animportant growth promoting gene was downregulatedin birds fed 2 mg of AFB1/kg. This could contributeto the observed reduction in growth rates of birds fedAFB1, and these results are consistent with earlier re-ports on the growth depressing effect of AF in broilerchicks (Huff et al., 1988).

Immune Response: Many genes associated withimmune function, such as mitogen-activated proteinkinase kinase 2, interleukin 10 receptor , ovomucoidprecursor, and interleukin 6 receptor were downregulat-ed, whereas tumor necrosis factor 10 was upregulatedin chicks fed 2 mg of AFB1/kg suggesting that AF isalso immunotoxic (Table 4). Previous reports suggestthat AF at dietary concentrations of 1 mg/kg or morecauses a significant reduction in the immune response(Verma et al., 2004). Similar reductions in immune re-sponse were also reported earlier in chicks fed 1 mgof AF/kg of diet (Shivachandra et al., 2003). Theseresults indicate an immunosuppressive effect of AF on

both humoral and cell-mediated immune responses.Because AF exerts part of its immunosuppressive ef-fects through cytokines (Han et al., 1999), downregula-tion of interleukins (interleukin 10 and interleukin 6)in the current study is consistent with earlier reports.Decreased immunity in chicks fed AF could make them

susceptible to secondary bacterial and viral infections,which could cause detrimental effects and even lead todeath. This hypothesis is supported by the increasedmortality rates that were observed in chicks fed AFB1in the present study.

Biotransformation, Detoxification, and Antioxi-dant Activity. Differentially expressed genes associatedwith biotransformation, detoxification, and antioxidantactivity are summarized in Table 5. Primary hepaticdetoxification processes include, xenobiotic biotrans-formation (phase I metabolism) and the subsequentconjugation of the resulting metabolites (phase II me-tabolism), making them more water soluble and avail-able for excretion from the body. Phase I metabolismmainly involves the cytochrome P450 (CYP) enzymes.Cytochrome P450 enzymes are associated with severalbiological interactions involving hydroxylation, epoxi-dation, dehydrogenation, nitrogen dealkylation, andoxidative deamination (Kumar et al., 2006). AlthoughCYP-mediated reactions are essential for xenobiotic

detoxification, they can also generate reactive oxygenspecies. The microsomal CYP-dependent mono-oxyge-nase system in the liver plays an essential role in themetabolism of xenobiotics (Akahori et al., 2005). Vari-ous CYP isoforms exist in species ranging from archae-bacteria to humans (Zuber et al., 2002). The CYP1A1

Table 4.Differentially expressed genes associated with immune system in B1-fed chicks compared with control birds at the end of21-d treatment period


Ratio, aflatoxin/control(log values)

FDR,1%

ENSGALT00000005554.1 Gal d I Ovomucoid precursor Immune response Down, 1.28 12.71ChEST691m10 IL10 Interleukin 10 receptor, Immune system Down, 1.28 12.53ENSGALT00000001934.1 MAPKK2 Mitogen-activated protein kinase kinase 2 Plays a critical role in the

production of cytokinesDown, 0.80 12.65

ggTRAIL TNFSF10 Tumor necrosis factor (ligand) superfamily,member 10 Tumor necrosis factorreceptor binding Up, 0.81 12.72

ChEST691m6 IL6 Interleukin 6 receptor Immune system Down, 2.28 12.00

1FDR = false discovery rate.

Table 5.Differentially expressed genes associated with biotransformation, detoxification, and antioxidant activities in aflatoxin B1-fed chicks compared with control birds at the end of 21-d treatment period1



FDR,%

ChEST332h21 IMP-3 Metalloproteinase inhibitor Endopeptidaseinhibitor activity Down, 1.07 12.57

ENSGALT00000027702.1 FDX1 Adrenodoxin mitochondrial precursor(Adrenal ferredoxin)


ChEST492f11 CYP2C45 Cytochrome p-450 2c45 Electron transport Down, 1.59 12.66ENSGALT00000020496.1 TGFB3 Heme oxygenase Antiinflammatory,

antioxidantUp, 0.70 12.80

ENSGALT00000008501.1 CYB5R NADH-cytochrome b5 reductase Electron transport Down, 1.13 12.53ChEST126e10 GST ,

LOC395611Glutathione s-transferase class- Antioxidant activity Down, 1.66 12.53

ChEST807d3 EH Epoxide hydrolase Detoxification Down, 0.57 12.77ChEST175g3 P24369 CYPB Peptidyl-prolyl cis-transisomerase Xenobiotic metabolism Up, 0.86 12.60X99454.1 CYP1A1 Cytochrome P450 1A1 Xenobiotic metabolism Up, 1.63 12.02M13454.1 CYP2H1 Cytochrome P450 2H1 Xenobiotic metabolism Up, 2.01 11.59NC0061002 GPx Glutathione peroxidase Xenobiotic metabolism Down, 1.59 10.63

1FDR = false discovery rate; NADH = nicotinamide adenine dinucleotide phosphate.



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is known to metabolize various drugs and xenobiotics(Klein et al., 2003), and is also known to activate cer-tain pro-mutagens to their carcinogenic forms (Haaset al., 2006; Kumar et al., 2006). Similarly, CYP 2H1is known to be actively involved in xenobiotic metabo-lism (Hamilton et al., 1993). These CYP isoforms areinvolved in biotransformation of AFB1 to the highlytoxic and carcinogenic metabolite AF-8, 9-epoxide inpoultry (Klein et al., 2003). Aflatoxin-8, 9- epoxideis detoxified by epoxide hydrolase (Tiemersma et al.,2001) and GST enzymes (Tiemersma et al., 2001; Kleinet al., 2003). Because CYP genes were upregulated andepoxide hydrolase and GST genes were downregulatedin birds fed 2 mg of AFB1/kg, there could be a greaterchance for formation of AF-8, 9-epoxide and less chancefor AFB1detoxification. Furthermore, overexpression of

these CYP450 isoforms was shown to induce chronicoxidative stress by generating more reactive oxygenspecies, possibly leading to hepatocellular injury anddeath (Kumar et al., 2006). It is evident from the re-sults of the present study that transcriptional activa-tion of CYP1A1 and CYP2H1 isoforms, in response toAF has the potential to increase oxidative stress. Fur-thermore, antioxidant genes such as GST and glutathi-one peroxidase, which could protect against oxidativestress, were downregulated in birds fed 2 mg of AFB1/kg, and this could further hamper the birds ability toprotect itself from oxidative damage. All of these fac-tors could contribute to the toxicological and pathologi-cal effects of AF.

Cell Proliferation. Cell proliferation is a representa-tion of cell growth and its active cell division. Aflatoxin

Table 6.Differentially expressed genes associated with cell proliferation in aflatoxin B1-fed chicks compared with control birds at theend of 21-d treatment period1


Ratio,aflatoxin/control(logvalues)

FDR,%

ChEST752c24 NR1D2 Nuclear receptor subfamily 1, groupd, member 2

Nucleobase, nucleoside, nucleotide, andnucleic acid metabolism

Up, 2.12 12.66

ENSGALT00000026260.1 DAX1 Adrenal hyoplasia protein Nucleobase, nucleoside, nucleotide, andnucleic acid metabolism Up, 0.44 12.79

ENSGALT00000006822.1 HDAC4 Histone deacetylase 4 Cell cycle progression Up, 0.71 12.79ENSGALG00000017034.1 FOXO1A forkhead box O1A

(rhabdomyosarcoma)Nucleic acid metabolism, transcription Up, 0.80 12.61

ENSGALG00000001139.1 FGFRL1 Fibroblast growth factor receptor Cell proliferation Up, 0.71 12.67NM_205187.1 SOX11 Sox11 transcription factor Transcription regulation Down, 1.19 12.68ENSGALT00000000605.1 Bach2 btb and cnc homology 2 Transcriptional regulator Down, 1.02 12.61ENSGALT00000001011.1 ATBF1 -fetoprotein enhancer-binding

proteinTranscription factor Up, 0.62 12.67

JUN v-jun sarcoma virus 17 oncogenehomolog (avian)

Transcription factor Up, 0.56 12.54

ENSGALT00000006994.1 TF9 Transcription factor sox-9 Transcription factor Up, 1.74 12.51ChEST752i6 SOX5 sry (sex determining region y)-box 5 Transcription Up, 1.40 12.56ChEST800g22 PPARA Peroxisome proliferator-activated

receptor Nucleic acid metabolism Up, 0.53 12.89

M_001012559.1 CDC2 Cell division cycle 10, g1 to s and g2

to m

Required for entry into S-phase and

mitosis in cell proliferation

Down, 0.86 12.59

ENSGALT00000024179.1 HDAC2 Histone deacetylase 2 Cell cycle progression and development. Up, 0.44 12.79ChEST372k22 DKK3 Dickkopf homolog 3 (Xenopus laevis) Inhibits Wnt signaling pathway Down, 3.50 11.80Contig_114_reverse FKBP1B fk506 binding protein 1b, 12.6 kda Cellular protein metabolism Down, 1.20 12.67ENSGALT00000017388.1 P50594 Protein mago nashi homolog mRNA splicing, nonsense mediated decay

pathwayDown, 0.55 12.52

ChEST90f21 PAPOLA Poly(a) polymerase Nucleic acid metabolism Down, 0.51 12.73ENSGALT00000026527.1 ODC1 Ornithine decarboxylase 1 Rate-limiting step in the pathway of

polyamine biosynthesisUp, 1.09 12.53

ENSGALT00000026837.1 FRZB Frizzled-related protein Regulate cell growth and differentiation Up, 0.98 12.51ENSGALT00000023380.1 LDB2 Lim domain binding 2 Transcription cofactor activity Down, 0.44 12.69ENSGALT00000021168.1 ESR1 Estrogen receptor (ER; estradiol

receptor)Cellular proliferation and differentiationin target tissues.

Up, 0.70 12.82

ENSGALT00000018678.1 NPM1 Ribonuclease Pyrimidine-specific nuclease withpreference for C

Up, 1.38 12.57

ChEST200a12 Cpeb3 Cytoplasmic polyadenylation elementbinding protein 3

Nucleotide-binding Up, 0.82 12.59

ENSGALT00000012486.1 NCL Nucleolin rRNA transcription and ribosomeassembly Down, 0.53 12.59

ENSGALT00000003436.1 NPM1 Nucleophosmin Nucleolar ribonucleoprotein structures Down, 0.54 12.56ENSGALT00000010377.1 UBE2I Ubiquitin-conjugating enzyme e2i

(ubc9 homolog, yeast)Covalent attachment of ubiquitin-likeprotein SUMO-1 to other proteins

Down, 0.76 12.65

ChEST200b24 TGFB3, Transforming growth factor, 3 Cellular morphogenesis, regulation of cellcycle, regulation of cell size

Down, 1.14 12.65


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B1 in the present study upregulated many genes as-sociated with cell proliferation, including peroxisomeproliferator-activated receptor , nuclear receptorsubfamily 1 d 2, bHLH transcription factor, ornithine

decarboxylase, histone deacetylase 2, transcription fac-tor sox-9, v-jun sarcoma virus 17 oncogene homolog,-fetoprotein enhancer-binding protein, and forkheadbox O1A (Table 6). Upregulation of the above men-tioned genes could contribute to increased cell prolif-eration rates in birds fed 2 mg of AFB1/kg. This couldbe the reason for hepatocarcinomas observed in rats fedAF (Kalengayi and Desmet, 1975; Butler and Hempsall,1981). Even though broilers do not generally live longenough to develop cancer, AF-related diseases adverse-ly affect their health and could cause slowing of growthand decreased resistance to microbial pathogens.

Blood Clotting. Genes involved in blood coagulation

[coagulation factor IX, coagulation factor X precursor(Stuart factor)] were downregulated, and anticoagulantprotein C precursor, which is an inactivator of coagula-tion factors Va and VIIIa, and antithrombin-III precur-

sor were upregulated in birds fed 2 mg of AFB1/kg (Ta-ble 7). Previous reports suggest that AF intoxicationwould lead to changes in coagulation profiles in lambsleading to an increased prothrombin time (Fernandez

et al., 1995). The authors suggested that prothrombintimes could be used as an indicator of aflatoxicosis inlambs. Increased clotting time was observed previouslyin chicks treated with single oral doses of AFB1 (50g/kg of BW; Obasi et al., 1994), and in chicks fed10.0 g/g of AF (Doerr and Hamilton, 1981). Bakerand Green (1987) stated that the coagulation defectin aflatoxicosis is primarily due to diminished hepaticsynthesis of coagulation factors. Clark et al. (1986) ob-served a significant decrease in factor IX, VIII, and Vactivities in AF-treated rabbits. They concluded thatthe coagulation factor deficiencies were the result ofdecreased factor synthesis due to hepatic insufficiency.

Bababunmi and Bassir (1982) found relatively low con-centrations of blood clotting factors II, VII, IX, and Xin the plasma of birds fed AF. Alteration in the expres-sion of the above-mentioned genes could be the reason

Table 8.Differentially expressed genes associated with cell skeletal structure in aflatoxin B1-fed chicks compared with control birdsat the end of 21-d treatment period1



FDR,%

ChEST471d22 Dnm1 Dynamin-1 Microtubule-associated force-producing protein Up, 0.76 12.72

ENSGALT00000008890.1 GJA1 Gap junction protein Form connexons between cells for lowmolecular weight material diffusion

Up, 0.99 12.77

ChEST148d1 LOC396480 Otokeratin Cytoskeleton Down, 2.7613.01

12.59

ENSGALT00000014968.1 KRT19 Keratin 19 Cytoskeleton Up, 0.6813.01

13.01

ENSGALT00000019859.1 arp3 Actin-related protein 3 homolog(yeast)

Cell motility Down, 0.58 12.97

ENSGALT00000019859.1 ACTA2 Actin, 2, smooth muscle, aorta Cell motility Down, 0.58 12.97ENSGALT00000009107.1 LOC395261 Filamin Actin binding Down, 1.26 12.61ENSGALT00000015293.1 RCJMB04_23c5 Actinin, 2 F-actin cross-linking bundling protein Down, 0.71 12.63ENSGALT00000018395.1 DCN Decorin Affect the rate of fibril formation Down, 0.48 12.55


Table 7.Differentially expressed genes associated with blood clotting in aflatoxin B1-fed chicks compared with control birds at theend of 21-d treatment period



FDR,1%

ENSGALT00000010110.1 HS2ST1 Heparan sulfate2-O-sulfotransferase

Blood coagulation Up, 0.70 12.75

ENSGALT00000009795.1 HS6ST2 Heparan sulfate6-O-sulfotransferase-2

Blood coagulation Up, 0.89 12.60

ENSGALG00000001780.11 PROC Anticoagulant protein Cprecursor Inactivator of coagulation factors vaand viiia Up, 0.94 12.77

ENSGALT00000010525.1 F9 Coagulation factor IX Blood coagulation Down, 1.29 11.36ENSGALG00000016833.1 VAP Coagulation factor X precursor

(Stuart factor)Blood coagulation Down, 0.81 12.52

ENSGALT00000010525.1 F9 Coagulation factor Blood coagulation Down, 1.29 11.36ENSGALG00000004591.1 ATIII Antithrombin-III precursor Generation of antithrombin-III Up, 0.57 12.87




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for the impaired coagulation in birds fed AF ultimatelyleading to hemorrhaging in the birds (Sandhu et al.,1995).

Cell Skeletal Structure. Nine genes associated withcell skeletal structure were differentially expressed inthe livers of chicks fed AFB1 (Table 8). Six of the 9genes were downregulated, and 3 were upregulated.The cytoskeleton of cells is a dynamic structure whose

functions include maintaining cell shape, cell protec-tion, enabling cellular motion, and it also plays im-portant roles in intracellular transport and cellular di-vision. Therefore, changes in the expression of genesassociated with cytoskeletal structure may negativelyaffect cell function. Ellinger-Ziegelbauer et al. (2004)hypothesized that changes in gene expression associatedwith cell structure organization may also be related tothe lesions observed in the hepatocytes of rats fed AF.Ellinger-Ziegelbauer et al. (2004) dosed rats with AFand observed an upregulation of several genes encodingproteins that function in cytoskeletal organization. Theauthors concluded that changes in the cytoskeleton are

likely both induced by and have an influence on thenecrotic process. As suggested by Ellinger-Ziegelbaueret al. (2004), changes in gene expression in the currentstudy may be an effort by cells to prevent cell necrosis,to assist in the regeneration of surrounding cells, orboth.

Current findings suggest that AFB1 at concentra-tions of 1 and 2 mg/kg has detrimental effects on feedintake, BW gain, and liver weights and adverse effectson serum TP, Ca, and P. Exposure of chicks to a 2 mgof AFB1/kg of diet results in physiological responsesassociated with altered gene expression in broiler chicklivers. However, future studies with pair fed birds will

determine whether the differential hepatic expressionof these genes is related to direct effects of AFB1or tosecondary effects on the liver associated with reducedfeed intake.

ACKNOWLEDGMENTS

I would like to thank Chada S. Reddy, associate pro-fessor, biomedical sciences, University of Missouri, Co-lumbia, MO, for his valuable suggestions. I also thankRoxanne Kutz, laboratory technician, Department ofAnimal Sciences, University of Missouri, Columbia,

MO for ordering materials and reagents required forthe study.

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