Effects of feeding corn naturally contaminated with aflatoxin B1

and B2 on hepatic functions of broilers

J. Yang,* F. Bai,*†1 K. Zhang,*1 S. Bai,* X. Peng,* X. Ding,* Y. Li,† J. Zhang,† and L. Zhao†

*Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry
of Education, Sichuan Agricultural University, Sichuan Ya’an 625014, China; and †Feed Products Quality
Monitoring Center of the Agricultural Ministry of China (Chengdu), Sichuan Chengdu 610041, China

ABSTRACT The purpose of this study was to evaluate increased (P < 0.05). The activity of superoxide dis-
the effects of feeding corn naturally contaminated with mutase and the content of hepatic malondialdehyde in-
aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) on serum creased when the broilers were fed with more than 50%
biochemical parameters, hepatic antioxidant enzyme contaminated corn (P < 0.05). A reduction in gluta-

activities, and pathological lesions of broilers. In total,
1,200 Cobb male broilers were randomly allocated into
5 treatments, with 8 replicates per treatment and 30
birds per replicate, in a 42-d experiment. The dietary
treatments were as follows: control, 25, 50, 75, and 100%
contaminated corn groups. Results showed that serum
aspartate aminotransferase activity in the 75 and 100%
contaminated groups were higher than that in the con-
trol group on d 21 (P < 0.05). Decreased content of
hepatic total protein and increased activities of hepatic
glutathione reductase and glutathione-S-transferase
were observed as the percentage of contaminated corn
Key words: aflatoxin B1, aflatoxin B2, broiler, antioxidant, pathological lesion
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 17, 2015
thione peroxidase level was observed in the AFB1- and
AFB2-contaminated groups on d 21 (P < 0.05). The
average pathological lesion scores and apoptosis rate
of liver cells increased as the concentration of dietary
AFB1 and AFB2 increased. Ultrastructural changes
were found in the livers of broilers fed 100% contami-
nated corn. In conclusion, diets containing AFB1 and
AFB2 could induce pathological lesions in the livers,
slightly change the serum biochemical parameters, and
damage the hepatic antioxidant functions when the in-
clusion of AFB1- and AFB2-contaminated corn reached
or exceeded 50%.
2012 Poultry Science 91:2792–2801
http://dx.doi.org/10.3382/ps.2012-02544

INTRODUCTION naturally contaminated grains varied with species, sex,

Mycotoxins are a group of secondary fungal metabo-
lites that occur widely in naturally contaminated foods
and feeds and have the most toxicity to animal health,
and they can cause significant financial losses to the
animal industries (Wu and Munkvold, 2008; Zhang and
Caupert, 2012).
It had been reported that grains naturally contami-
nated with fusarium (9.5~12.1 mg/kg of deoxynivale-
nol, DON) reduced performance and immune function,
and altered intestinal morphology, hematology, and se-
rum chemistry in broilers (Swamy et al., 2002, 2004;
Chowdhury and Smith, 2004; Awad et al., 2006). Other
researchers observed, however, that broilers had a high
tolerance to fusarium (38~82.8 mg/kg of DON) con-
tained in naturally contaminated grains (Lun et al.,
1986; Moran et al., 1987). The response to toxicity of
©2012 Poultry Science Association Inc.
Received June 16, 2012.
Accepted July 8, 2012.
1 Corresponding authors: baifan-111@hotmail.com and zkeying@
yahoo.com
age, and health status of broilers and the concentration
and length of exposure to mycotoxins (Jewers, 1990).
The primary mycotoxins of concern in poultry feed-
stuffs are aflatoxins, mainly including 4 major forms:
aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin
G1 (AFG1), and aflatoxin G2 (AFG2; Monbaliu et
al., 2010). The order of toxicity is AFB1 > AFG1 >
AFB2 > AFG2, among which AFB1 is generally the
most common one in feedstuffs (Fandohan et al., 2005).
Many studies have demonstrated that feeding purified
AFB1 has an adverse effect on the growth of broilers
(Ledoux et al., 1999; Hashmi et al., 2006; Kermanshahi
et al., 2007; Yarru et al., 2009; Magnoli et al., 2011).
Liver is the main target organ of aflatoxins, and histo-
pathological examination by optical microscope can be
used as an effective method for diagnosis of aflatoxico-
sis (Ellakany et al., 2011). Previous studies have dem-
onstrated the hepatotoxic effect of higher AFB1 (1–5
mg/kg) on broilers, leading to pathological lesions of
the livers (Rosa et al., 2001; Eraslan et al., 2006; Ku-
mar and Balachandran, 2009). However, few literature
studies have examined the effect of grains naturally

2792
 INFLUENCE OF AFLATOXINS ON THE LIVER OF BROILERS 2793
Table 1. Composition and nutrient levels of the basal diets (%, as-fed basis)
Diet (%)

Item 1 to 21 d 22 to 42 d

Ingredient    
 Corn 60.00 63.00
  Soybean meal (46%) 31.20 28.17
  Corn gluten meal 3.00 2.50
  Soybean oil 1.14 2.27
  Calcium carbonate 0.95 0.88
  Calcium hydrophosphate 1.85 1.60
  l-Lysine hydrochloride 0.08 0.02
  dl-Methionine 0.16 0.09
  Sodium chloride 0.50 0.40
  Choline chloride 0.15 0.10
  Vitamin and mineral premix 0.331 0.322
  Rice hull 0.64 0.65
 Total 100.00 100.00
Chemical composition
 CP 20.83 19.37

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 ME,3 MJ/kg 12.15 12.55
 Calcium 0.97 0.87
  Available phosphorus 0.44 0.39
 Lysine 1.11 0.97
 Methionine 0.48 0.39
  Methionine + cystine 0.86 0.72
 Threonine 0.78 0.74
1Provided per kilogram of diet: vitamin A, 12,000 IU; cholecalciferol, 3,000 IU; vitamin E, 7.5 IU; vitamin K ,
3
1.5 mg; thiamine, 0.6 mg; riboflavin, 4.8 mg; pyridoxine, 1.8 mg; vitamin B12, 9 μg; folic acid, 150 μg; niacin, 10.5
mg; calcium pantothenate, 7.5 mg; iron, 100 mg; copper, 8 mg; manganese, 120 mg; zinc, 100 mg; selenium, 0.3
mg; iodine, 0.7 mg.
2Provided per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 2,000 IU; vitamin E, 5 IU; vitamin K , 1
3
mg; thiamine, 0.4 mg; riboflavin, 3.2 mg; pyridoxine, 1.2 mg; vitamin B12, 6 μg; folic acid, 100 μg; niacin, 7 mg;
calcium pantothenate, 5 mg; iron, 80 mg; copper, 8 mg; manganese, 100 mg; zinc, 80 mg; selenium, 0.3 mg; iodine,
0.7 mg.
3The ME of the diet was calculated according to NRC (1994).

contaminated with low levels of aflatoxins on the health
of broilers (McKenzie et al., 1998; Aravind et al., 2003;
Ghahri et al., 2010).
Although results from our previous study demonstrat-
ed that feeding corn naturally contaminated with AFB1
and AFB2 did not affect apparent nutrient digestibility
of broilers, it decreased performance and altered small
intestinal morphology (J. Yang, unpublished data).
Whether the poor performance was caused by the ef-
fect of contaminated corn on other functions needs fur-
ther study. Thus, this study was conducted to evaluate
the effects of feeding corn naturally contaminated with
AFB1 and AFB2 on the hepatic functions of broilers.
MATERIALS AND METHODS
Experimental Design and Diets
The bird experiment was conducted in accordance
with guidelines approved by Animal Health and Care
Committee of Sichuan Agricultural University.
The experiment was conducted using a single facto-
rial experiment design, and the proportions of naturally
contaminated corn to substitute for normal corn were
0, 25, 50, 75, and 100% in the diets, respectively. Each
treatment had 8 replicates, with 30 birds per replicate.
The basal diet was based on corn and soybean meal,
with composition and nutrient levels in line with NRC
(1994) recommendations for broilers (Table 1). All the
ingredients in the diets except the corn were the same.
Birds and Management
In total, 1,200 one-day-old male Cobb broiler chicks
from a commercial hatchery were weighed and ran-
domly distributed into 5 treatments. The experiment
lasted for 6 wk, consisting of a starter phase from d 0
to 21 and a grower phase from d 22 to 42. Birds were
raised in cage pens at the Animal Nutrition Institute of
Sichuan Agricultural University in China. Chicks were
maintained on a 24-h continuous light schedule. The
temperature was initially maintained at 33 ± 1°C and
gradually reduced by 3°C per week to reach 21°C after
35 d, and this temperature was maintained for the du-
ration of the experiment. Feed and water were provided
ad libitum.
Corn and Dietary Mycotoxin Concentrations
Corn and feed samples were ground in a Retsch
ZM100 mill (Retsch, Newtown, PA) using a sieve mea-
suring 0.75 mm and were analyzed to determine the
content of mycotoxins, including AFB1, AFB2, AFG1,
AFG2, T-2 toxin, DON, zearalenone (ZEA), ochratox-
in A (OTA), and fumonisin B1 (FB1). Briefly, myco-
toxins were extracted from 25 g of milled sample with
2794 Yang et al.
50 mL of water for DON; an acetonitrile and water so-
lution (90:10, vol/vol) for ZEA; a methanol and water
solution (70:30, vol/vol) for aflatoxins; and a metha-
nol and water solution (80:20, vol/vol) for T-2 toxin,
OTA, and FB1. The mixtures were shaken vigorously
for 40 min. The extract was filtered, and then 10 mL
of the filtrate was mixed with 40 mL of PBS. The mix-
ture was centrifuged at 8,000 × g for 15 min. A 15-mL
quantity of the supernatant was then applied onto the
corresponding mycotoxin immunoaffinity column (Ro-
man Corporation, Red Hill, Singapore). The column
was washed with 10 mL of PBS and 10 mL of water and
then eluted with 2 mL of methanol into a glass vial,
which was maintained at 4°C until analysis.
The mycotoxins were quantified via an HPLC instru-
ment equipped with a fluorescence detector (Agilent
1100, Agilent Technologies, Santa Clara, CA). Separa-
tion was achieved on an SB-C18 column (4.6 × 250
mm; 5 μm; Agilent Technologies). The mobile phases
were mixtures of a methanol and water solution (45:55,
vol/vol) for aflatoxins; an acetonitrile and water solu-
tion (12:88, vol/vol) for DON; an acetonitrile and water
solution (80:20, vol/vol) for T-2 toxin; an acetonitrile,
water, and methanol solution (46:46:8, by vol) for ZEA;
a methanol and aqueous 1% acetic acid solution (50:50,
vol/vol) for OTA; and a solution of methanol and water
acidified with 0.1 mol/L of phosphoric acid (pH 3.3;
50:50, vol/vol) for OTA. The flow rate was 1 mL/min,
and the injection volume was 50 μL.
Postcolumn photochemical derivatization was used
to enhance the mycotoxin response using a PHRED
photochemical reactor (AURA Industries, New York,
NY). The excitation and emission wavelengths were
fixed at 365 and 440 nm for aflatoxins, 330 and 460 nm
for OTA, 330 and 440 nm for FB1, and 381 and 470
nm for T-2 toxin, respectively. However, the presence
of ZEA and DON was monitored at 274 and 220 nm,
respectively, with the fluorescence detector. The detec-
tion limits of the above mycotoxins were 2 μg/kg for
AFB1, 0.8 μg/kg for AFB2, 2.5 μg/kg for AFG1, 1.5
μg/kg for AFG2, 100 μg/kg for T-2 toxin, 300 μg/kg
for DON, 100 μg/kg for ZEN, 30 μg/kg for OTA, and
200 μg/kg for FB1 (AQTSSP, 2010).
Serum Biochemistry and Organ Weights
On d 22 and 43, one bird per replicate, for a total of
8 birds per treatment, was selected for blood sampling
via the jugular vein; no anticoagulant was used. Blood
samples were centrifuged at 900 × g for 10 min at 4°C
to obtain sera, which were then stored at −20°C for the
determination of serum biochemical parameters. Se-
rum total protein (TP); albumin (ALB) content; ala-
nine transaminase (ALT), aspartate aminotransferase
(AST), and γ-glutamyltransferase (γ-GT) enzyme ac-
tivities were measured by the colorimetric method. The
specific assay kits were purchased from the Nanjing Ji-
ancheng Bioengineering Institute (Nanjing, China). Af-
ter blood collection, chicks were euthanized humanely.
The livers were removed and weighed [data expressed
as relative liver weight (g of liver/100 g of BW)].
Hepatic Antioxidant Enzyme Activities
On d 22 and 43, another bird from each replicate of
each treatment was euthanized, and liver samples (at
the same part) were obtained and then stored at −20°C
until laboratory analyses were conducted. Liver sam-
ples (about 0.5 g) and physiological saline were mixed
at a ratio of 1:9 to make tissue homogenates, and the
homogenates were then centrifuged at 1,200 × g for 10
min at 4°C to obtain a supernatant fluid. The superna-
tant fluid contents of TP, malondialdehyde (MDA), ac-
tivities of glutathione reductase (GR), glutathione per-
oxidase (GSH-Px), glutathione-S-transferase (GST),
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superoxide dismutase (SOD), and total antioxidant ca-
pacity were measured by the colorimetric method. The
specific assay kits were purchased from the Nanjing Ji-
ancheng Bioengineering institute of China.
Pathological Lesions and Apoptosis
On d 22 and 43, liver samples were fixed in 4% buff-
ered formaldehyde and routinely embedded in paraffin.
Thin sections (5 μm) were sliced and mounted on a
slide, and then stained with hematoxylin and eosin for
histopathological examination by an Olympus CX31-
32C02 optical microscope (Olympus Optical Company,
Tokyo, Japan). According to the severity of hyperplastic
bile duct epithelium, the severity of lesions was scored
subjectively as follows: 0, normal histological structure;
1, funicular hyperplasia of the bile duct epithelium was
recognizable around some portal areas; 2, funicular hy-
perplasia of the bile duct epithelium was observed in
almost all portal areas, and the region of hyperplasia
was approximately half of the hepatic lobule; 3, funicu-
lar hyperplasia of the bile duct epithelium was involved
in the whole hepatic lobule, and showed as a reticular
pattern; 4, the bile duct epithelium presented as diffuse
hyperplasia, and much more massive hyperplasia of the
bile duct epithelium was observed in portal areas.
On d 43, one bird each from the control and 100%
contaminated groups was randomly selected. Livers
were dissected and then fixed in 2.5% glutaraldehyde
and postfixed in 2% veronal acetate-buffered OsO4. Af-
ter dehydration in graded alcohol, they were embedded
in Araldite (Huntsman Advanced Materials LLC, Salt
Lake City, UT). The sections, about 70 nm thick, were
stained with uranyl acetate, poststained with 0.2% lead
citrate, and examined with an H-600 electron micro-
scope (Hitachi Company, Tokyo, Japan).
The apoptosis rate of liver cells was determined ac-
cording to the method described previously (Peng et al.,
2009). Briefly, representative tissues selected from the
livers were made into cell homogenates, passed through
a 0.05-mm screen, and centrifuged at 25 to 60 × g at
4°C for 5 min. After the supernatants were discarded,
the precipitates were washed 2 times with PBS (pH 7.0
 INFLUENCE OF AFLATOXINS ON THE LIVER OF BROILERS 2795
Table 2. Concentrations of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in the diet and corn (μg/
kg)
Normal Contaminated
Diet Control 25% 50% 75% 100% corn corn

1 to 21 d
 AFB1 ND1 16.3 36.9 45.6 82.4 ND 149.6
 AFB2 ND 3.2 6.4 7.9 14.2 ND 24.2
22 to 42 d
 AFB1 ND 34.3 69.3 95.2 134.0 ND 229.0
 AFB2 ND 6.2 12.1 17.0 23.6 ND 37.8
1ND = not detectable.

to 7.4). The single-cell suspensions were obtained and ZEA, OTA, T-2 toxin, and FB1) were below the limit
their concentrations were adjusted to 1 × 106 cells/mL. of detection.
After that, the 100-μL cell suspensions were placed in
a flow tube and 5 μL of Annexin V fluorescein isothio- Organ Weight and Serum Biochemistry

cyanate and 5 μL of polytrans isoprene were added.
The flow tube was mixed by shaking slightly and kept
standing for 15 min away from light at room tempera-
ture. Then, 400 μL of binding buffer was added and
mixed. The apoptosis rate of livers was determined by
flow cytometry and analyzed by Cell Quest software
(Becton Dickinson and Co., Franklin Lakes, NJ).
Statistical Analysis
Data were analyzed by one-way ANOVA using SPSS
11.0 (SPSS Inc., Chicago, IL). When the ANOVA
showed significance, Duncan’s significant-difference
test was applied. All statements of differences were
based on a significance level of P < 0.05.
RESULTS
Dietary Mycotoxin Concentrations
The naturally contaminated corn used in these di-
ets was mainly contaminated with AFB1 and AFB2.
The concentrations of AFB1 in the experimental diets
ranged from 16.3 to 82.4 μg/kg in the starter period
and from 34.3 to 134.0 μg/kg in the grower period. The
contents of AFB2 in diets ranged from 3.2 to 14.2 μg/
kg and from 6.2 to 23.6 μg/kg in the starter and grower
periods, respectively. The contents of AFB1 and AFB2
were different because of different storage times and
contamination degrees of corn (Table 2). The contents
of other mycotoxins (including AFG1, AFG2, DON,
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As shown in Table 3, as the ratio of contaminated
corn increased, the BW of broilers decreased. Com-
pared with the control, the BW of broilers in the 100%
contaminated group decreased significantly. There was
no influence on the BW of broilers on d 42. No differ-
ence was shown in the absolute and relative weights of
livers among treatments on d 21. However, the abso-
lute weight of liver in the 25% contaminated group was
lower (P < 0.05) than that in other groups on d 42, and
the relative weight of liver in the 25% contaminated
group was lower (P < 0.05) than that in the 50, 75, and
100% contaminated groups on d 42.
The results of serum biochemical parameters are giv-
en in Table 4. On d 21, compared with other groups,
the activity of serum AST increased (P < 0.05) when
birds were fed 75 and 100% contaminated corn. Se-
rum γ-GT activity in the 100% contaminated group
was higher (P < 0.05) than that in the 25 and 50%
contaminated groups, whereas it was higher (P < 0.05)
in the 75% contaminated group than in the 25% con-
taminated group. There was no difference in contents of
TP, ALB, and ALT on d 21 or in TP, ALB, ALT, AST,
and γ-GT concentrations on d 42.
Hepatic Antioxidant Enzyme Activities
On d 21, compared with the control, the hepatic pro-
tein contents were decreased (P < 0.05) when the level
of contaminated corn was increased (P < 0.05), with a
significant difference in the 50 or 100% contaminated

Table 3. Effects of corn naturally contaminated with aflatoxin B1 and aflatoxin B2 on relative liver weights of broilers
Item Control 25% 50% 75% 100% SEM P-value

21 d
  BW (g) 822.5ab 839.0a 803.4b 794.5b 749.7c 6.558 <0.001
  Absolute liver weight (g) 18.1 18.0 18.9 18.4 18.1 0.291 0.874
  Relative liver weight (g/100 g of BW) 2.19 2.15 2.40 2.32 2.42 0.041 0.14
42 d
  BW (g) 2,683.5 2,533.0 2,618.3 2,403.3 2,488.0 47.437 0.356
  Absolute liver weight (g) 72.2a 56.6b 76.7a 71.4a 76.7a 2.324 0.026
  Relative liver weight (g/100g of BW) 2.68ab 2.29b 2.96a 2.97a 2.97a 0.076 0.006
a–cDifferent lowercase superscripts in the same row indicate a significant difference between treatments (P < 0.05).
2796 Yang et al.
Table 4. Effects of corn naturally contaminated with aflatoxin B1 and aflatoxin B2 on serum biochemical parameters of broilers1
Item Control 25% 50% 75% 100% SEM P-value

21 d
  TP (g/L) 23.16 20.02 20.75 22.55 22.89 0.676 0.504
  ALB (g/L) 13.93 11.82 11.37 12.85 11.72 0.390 0.245
  ALT (U/L) 3.12 2.81 3.05 2.89 3.83 0.189 0.496
  AST (U/L) 19.63b 19.25b 20.03b 22.53a 23.41a 0.452 0.004
  γ-GT (U/L) 22.36abc 18.81c 21.75bc 25.08ab 27.94a 0.950 0.02
42 d
  TP (g/L) 27.05 28.49 25.02 26.74 25.28 0.751 0.591
  ALB (g/L) 25.29 20.63 22.56 15.81 21.66 1.224 0.166
  ALT (U/L) 3.08 2.96 3.61 3.76 3.32 0.171 0.536
  AST (U/L) 25.59 23.17 22.62 25.42 26.86 0.669 0.219
  γ-GT (U/L) 32.75 28.81 34.05 33.35 35.08 1.085 0.382
a–cDifferent lowercase superscripts in the same row indicate a significant difference between treatments (P < 0.05).
1TP = total protein; ALB = albumin; ALT = alanine transaminase; AST = aspartate aminotransferase; γ-GT = γ-glutamyltransferase.

groups (Table 5). The same trend was found for GSH- hyperplasia of the bile duct epithelium became obvious

Px activities, with a decrease (P < 0.05) in the 25, 75,
and 100% contaminated groups; GST activities in all
contaminated groups were increased (P < 0.05), and
the same trend was found for GR, with a significant
difference (P < 0.05) in the 75 and 100% contaminated
groups. The MDA content and SOD activity were in-
creased (P < 0.05) in the 50% contaminated group.
On d 42, all the contaminated groups resulted in a
reduction in hepatic protein (P < 0.05). The contents
of GR, GST, and SOD were numerically increased with
increased levels of contaminated corn, but a significant
difference (P < 0.05) was found only in the 75% and
100% contaminated groups for GR and GST contents
and in the 100% contaminated group for SOD level.
The MDA content in the 75% contaminated group
showed an increase compared with that of the control
group (P < 0.05). No effect on GSH-Px activities was
observed on d 42 and on total antioxidant capacity on
d 21 and 42 among treatments.
Pathological Lesions and Apoptosis
In the control group, all livers had a normal histologi-
cal structure (scores 0; Figures 1a and 1b). On d 21,
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as the contaminated corn increased, and the average
scores in the 25, 50, 75, and 100% contaminated groups
were 0.67, 0.83, 1.83, and 3.67, respectively (Table 6).
In the 25 and 50% contaminated groups, few hyperplas-
tic bile duct epithelia were observed (score 1; Figure 1c
and 1d). In the 75% contaminated group, 83.3% (5/6)
of the livers had light lesions (scores 1 and 2; Figure
1e and 1f), and 16.7% (1/6) of livers had severe lesions
(score 4; Figure 1i and 1j). In the 100% contaminated
group, all the livers had severe lesions; 33.3% (2/6) of
the livers were scored 3 (Figure 1g and 1h) and the rest
(4/6) were scored 4 (Figure 1i and 1j). On d 42, histo-
pathological lesions of livers in the AFB1- and AFB2-
contaminated groups were all alleviated; the average
scores in the control, 25, 50, 75, and 100% contami-
nated groups were 0, 0, 0.17, 0.33, and 0.5, respectively.
Ultrastructural changes were observed in the 100%
contaminated group compared with the control group
(Figure 2). Hepatocytes showed fatty degeneration; dif-
ferently sized lipid droplets presented in the cytoplasm.
The endoplasmic reticulum was swollen and many
secondary lysosomes were present in the cytoplasm.
Karyomorphism became irregular, and swelling of the

Table 5. Effects of corn naturally contaminated with aflatoxin B1 and aflatoxin B2 on hepatic antioxidant enzymes of broilers1
Item Control 25% 50% 75% 100% SEM P-value

21 d
  Liver protein (mg/100 mg of tissue) 8.21ab 8.72a 7.28c 7.59bc 6.96c 0.156 <0.001
  MDA (nmol/mg of protein) 0.36bc 0.48b 0.69a 0.30c 0.39bc 0.029 <0.001
  T-AOC (U/mg of protein) 2.52 2.56 2.34 2.67 2.39 0.053 0.299
  GR (U/g of protein) 7.26c 7.83bc 8.78abc 9.40ab 10.00a 0.325 0.04
  GST (U/mg of protein) 24.13c 27.17b 29.29ab 30.38a 28.67ab 0.554 0.001
  GSH-Px (U/mg of protein) 51.14a 42.34bc 45.10ab 40.22bc 37.39c 1.262 0.003
  SOD (U/mg of protein) 210.57b 219.83b 261.93a 223.22b 214.63b 4.293 0.001
42 d
  Liver protein (mg/100 mg of tissue) 8.56a 7.72b 7.56bc 6.94c 6.86c 0.143 <0.001
  MDA (nmol/mg of protein) 0.36b 0.56ab 0.39b 0.60a 0.44ab 0.031 0.057
  T-AOC (U/mg of protein) 2.35 2.37 2.36 2.18 2.27 0.069 0.903
  GR (U/g of protein) 7.48c 8.37bc 9.03abc 10.68a 10.25ab 0.336 0.008
  GST (U/mg of protein) 31.32b 32.62b 35.15ab 38.37a 38.55a 0.814 0.006
  GSH-Px (U/mg of protein) 47.25 49.60 44.57 49.42 43.44 0.878 0.072
  SOD (U/mg of protein) 244.46b 250.92b 250.49b 264.19b 292.55a 4.447 0.001
a–cDifferentlowercase superscripts in the same row indicate a significant difference between treatments (P < 0.05).
1GR = glutathione reductase; SOD = total superoxide dismutase; GST = glutathione-S-transferase; MDA = malondialdehyde; T-AOC = total
antioxidant capacity; GSH-Px = glutathione peroxidase.
 INFLUENCE OF AFLATOXINS ON THE LIVER OF BROILERS 2797
Table 6. Effects of corn naturally contaminated with aflatoxin B1 and aflatoxin B2 on liver histopa-
thology of broilers1
Item Control 25% 50% 75% 100%

21 d
 0 6 2 1 0 0
 1 0 4 5 3 0
 2 0 0 0 2 0
 3 0 0 0 0 2
 4 0 0 0 1 4
 Mean2 0 0.67 0.83 1.83 3.67
42 d
 0 6 6 5 4 3
 1 0 0 1 2 3
 2 0 0 0 0 0
 3 0 0 0 0 0
 4 0 0 0 0 0
 Mean2 0 0 0.17 0.33 0.5
1Six samples were used to evaluate the histopathological score.
2The mean was calculated by the addition of different scores multiplied by the corresponding ratio of sick broil-

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ers.

nuclear membrane was observed. Some circular-shapes
structures appeared in the nucleus.
The data given in Table 7 indicate that as the level of
contaminated corn increased, the apoptosis rate of liver
cells increased on d 21 (P < 0.05) and d 42 (P = 0.070).
Compared with the control, significant increases were
found in the 50, 75, and 100% contaminated groups on
d 21 (P < 0.05) and in the 75 and 100% contaminated
groups on d 42 (P < 0.05).
DISCUSSION
In this study, the BW of broilers selected for deter-
mination of the relative weight of livers was close to the
average BW of the corresponding replicate. Hence, the
result was not completely similar to other data on per-
formance (J. Yang, unpublished data). Birds fed corn
naturally contaminated with AFB1 and AFB2 tended
to have an increase in the relative weight of livers that
was similar to the results from other researchers (Mi-
azzo et al., 2000; Teleb et al., 2004; Sakhare et al., 2007;
Safameher, 2008). The inhibition of lipid transport may
result in lipid accumulation and the formation of a spe-
cific and enlarged fatty liver (Tung et al., 1972). Our
study failed to observe any statistical change in the
relative weight of livers, which may have been caused
by the low levels of AFB1 and AFB2 in the diets. The
mechanism explaining why the absolute and relative
weights of livers in the 25% contaminated group were
lower than those in the other contaminated groups on d
42 is not clear. It may be due to the stimulating effect
of low levels of dietary AFB1 and AFB2.

Figure 1. Morphology of chicken livers from the control and aflatoxin B1-contaminated groups on d 21 and d 42. (a) Control (0 points),
hematoxylin and eosin (H.E) 200×; (b) control, cytoplasm is homogeneous and less connective tissue is present in the portal areas (0 points),
H.E 400×; (c) slight hyperplastic bile duct epithelium is present in some portal areas (1 points), H.E 200×; (d) bile duct epithelia with funicular
hyperplasia are present in the portal areas (1 points), H.E 400×; (e) hyperplastic bile duct epithelia are observed in the portal areas, and the
regions are approximately half of hepatic lobule (2 points), H.E 200×; (f) bile duct epithelia with funicular hyperplasia are present (2 points), H.E
400×; (g) hyperplasia of the bile duct epithelium is involved in the whole hepatic lobule (3 points), H.E 200×; (h) hyperplastic bile duct epithelia
are involved in major areas (3 points), H.E 400×; (i) bile duct epithelium with diffuse hyperplasia is present and hepatic tissues are damaged (4
points), H.E 200×; (j) massive hyperplasia of the bile duct epithelium is observed in portal areas (4 points), H.E 400×. Color version available
in the online PDF.
2798 Yang et al.
Table 7. Effects of corn naturally contaminated with aflatoxin B1 and aflatoxin B2 on hepatic apop-
tosis rate of broilers
Item Control 25% 50% 75% 100% SEM P-value

21 d 4.07c 4.82c 6.31b 6.38b 8.17a 0.313 <0.001

42 d 4.76b 5.11ab 5.40ab 6.04a 6.04a 0.168 0.070
a–cDifferent lowercase superscripts in the same row indicate a significant difference between treatments (P <
0.05).

When hepatocyte permeability increased or hepato-
cytes were damaged, transaminase may have been re-
leased from hepatocytes into the blood and increased
serum transaminase activity. The reduced serum total
protein was probably due to the inhibition of amino
acid transport and mRNA transcription, resulting in
the inhibition of DNA and protein synthesis (Thaxton
kg) significantly decreased serum TP content and ALT
and AST activities of broilers on d 21 and had no effect
on these serum indices on d 35 (Aravind et al., 2003).
These differences were due to naturally contaminated
corn containing complex ingredients, which was differ-
ent from purified AFB1. Furthermore, the species, age
of bird, dosage, and time of mycotoxin exposure could

et al., 1974). The present study showed that the 75 and
100% contaminated corn significantly increased serum
AST activity and had an increased tendency for γ-GT
compared with the control on d 21. This result was in
agreement with previous studies on broilers (Bintvihok
and Kositcharoenkul, 2006; Ghahri et al., 2010) and on
laying hens (Fernandez et al., 1994), illustrating that
aflatoxin-contaminated corn can cause damage to the
hepatocytes of broilers and increased serum transami-
nase activities. Furthermore, no significant difference
was observed in serum TP and ALB contents and ALT
enzyme activity, which agreed with the results of some
studies on broilers (Fernandez et al., 1994; Tedesco et
al., 2004). However, other reports indicated that AFB1
(0.1~5 mg/kg) significantly improved serum ALT and
AST activities and decreased TP and ALB contents
(McKenzie et al., 1998; Oğuz et al., 2000; Cao et al.,
2010). Aravind reported that grains naturally contami-
nated with mycotoxins (aflatoxins, 168 μg/kg; ochra-
toxin, 8.4 μg/kg; ZEA, 54 μg/kg; T-2 toxin, 32 μg/
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 17, 2015
not be ignored.
Under normal physiological conditions, the organism
maintains a dynamic balance between the oxidative
system and the antioxidant system. Pathological fac-
tors and toxic materials may break down this balance,
accumulate free radicals, and induce oxidative damage.
Enzymes such as SOD, GSH-Px, GST, and GR are
crucial components of the antioxidant system and play
a key role in removing oxygen free radicals and reliev-
ing oxidative damage. Our study indicated that diets
containing AFB1 and AFB2 significantly reduced he-
patic protein content and increased hepatic GST and
GR activities, a finding supported by other reports
(Gawai et al., 1992; Quezada et al., 2000; Valdivia et
al., 2001). The toxic damage of AFB1 depends on its
second metabolite of AFB1-8,9-expoxide. The GST cat-
alyzse AFB1-8,9-expoxide to covalently bind with re-
duced glutathione and eliminates toxins from the body
as AFB1-mercaptan uric acid (Moss et al., 1985; Raney
et al., 1992). The GR is mainly responsible for convert-

Figure 2. Ultrastructural changes of hepatocytes from broilers that were fed a control diet or a 100% aflatoxin B1-contaminated diet for 42
d. (a) Control, with a normal nucleus and organelles; (b–f) 100% aflatoxin B1-contaminated group; (b) fatty degeneration, lipid droplets with
variable sizes appeared in the cytoplasm (6,000×); (c) swelling of the endoplasmic reticulum, irregular nuclei (8,000×); (d) many more secondary
lysosomes are present in cytoplasm (10,000×); (e) swelling of nuclear membrane (12,000×); (f) circular chromatin (17,000×).
 INFLUENCE OF AFLATOXINS ON THE LIVER OF BROILERS
ing oxidized glutathione into reduced glutathione, and
increased GR activity means that reduced glutathione
is increased. The increases in GST and GR activities
demonstrated that under the stimulation of low AFB1
levels, broilers met the requirement of detoxification by
secreting much more GST and GR. Reduced hepatic
protein content was caused by inhibition of protein syn-
thesis due to the damage of the liver. The SOD plays a
vital role in the conversion of O2− into H2O2, and GSH-
Px then converts H2O2 into H2O. As the main product
of lipid peroxidation, MDA is an important index of an-
tioxidant ability (Wills, 1966). In our study, corn natu-
rally contaminated with AFB1 and AFB2 significantly
enhanced the hepatic MDA content, decreased GSH-Px
activity (d 21), and had no effect on total antioxidant
ability. Similar results were observed in previous studies
(Shi et al., 2006; Gowda et al., 2008; Hou et al., 2008),
which suggested that aflatoxins could induce hepatic
lipid peroxidation and damage the antioxidant system.
The GSH-Px activity showed no difference on d 42,
which may be attributed to the increased tolerance of
broilers. However, it is interesting that we found AFB1
and AFB2 contaminated corn treatments enhanced
the hepatic SOD activity, which was in contrast to the
studies mentioned above. This discrepancy may be as-
cribed to unknown ingredients contained in naturally
contaminated corn or the body having a response to
low levels of AFB1 and AFB2 to produce more SOD for
the removal of increased oxygen free radicals by adjust-
ing itself. Regretfully, we failed to establish a significant
dose-effect relationship between treatments for MDA,
GSH-Px, and SOD, for which we thought the difference
in individuals and the sensitivity to low levels of AFB1
and AFB2 were important.
No macroscopic changes in liver was found at low
levels (50 to 100 μg/kg) of aflatoxins (Ortatatli et al.,
2005). However, other researchers reported that lower
aflatoxins (30 to 200 μg/kg) induced hepatic architec-
ture enlargement, fatty degeneration, bile-duct hyper-
plasia, periportal fibrosis, hepatocytic vacuolation, and
necrosis through microscopic investigation (Teleb et
al., 2004; Ortatatli et al., 2005; Ellakany et al., 2011),
which was consistent with the present study. The al-
terations in the ultrastructure of hepatocytes observed
in the 100% contaminated group also clarified that corn
naturally contaminated with AFB1 and AFB2 caused
pathological lesions of the hepatocytes. We also found
that the degree of damaged livers increased with in-
creasing levels of contaminated corn. Furthermore, the
extent of damage on d 21 was greater than that on d
42, which was probably due to the enhanced tolerance
to aflatoxins as the broilers aged.
Apoptosis is a specialized process of cell death that
is part of the normal development of organs and tissue
maintenance, but it may also occur as a response to var-
ious environmental stimuli, indicating toxicity (Ribeiro
et al., 2010). In the present study, corn naturally con-
taminated with AFB1 and AFB2 significantly increased
the rate of hepatic apoptosis, which agreed with the
2799
results of studies on male rats (Meki et al., 2001) and
on ducks (He, 2011). The apoptosis result also demon-
strated that the AFB1- and AFB2-contaminated corn
damaged the hepatic tissues at the molecular level.
Aflatoxin B1 is mainly metabolized in the liver and
its second metabolite of AFB1-8,9-expoxide can com-
bine with DNA, cause canceration of hepatic cells, and
damage the hepatic functions thereafter (Swenson et
al., 1977; Yoshizawa et al., 1982; Eaton and Gallagher,
1994). Through the observation of pathological changes
and determination of the rate of apoptosis, we found
that corn naturally contaminated with AFB1 and AFB2
damaged the hepatic tissue, and then transaminase was
released into the blood and caused serum transaminase
activities to be enhanced, but this effect was not obvi-
ous, with the only changes in AST and γ-GT activi-
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 17, 2015
ties occurring on d 21. We hypothesize that feeding
aflatoxins resulted in the inhibition of hepatic protein
synthesis and lipid metabolism, including a decrease
in hepatic protein content, the formation of fatty liver,
and an increase in the relative weight of livers. The
reduced antioxidant functions in the current study indi-
cated that aflatoxins caused hepatic lipid peroxidation
and damaged the antioxidant system. This may have
resulted from the mechanisms of cell and DNA dam-
age induced by aflatoxins (Shen et al., 1994). The im-
pairments of hepatic functions and metabolism induced
by AFB1- and AFB2-contaminated corn were probably
responsible for reduced performance of broilers. More-
over, the toxic effects of corn naturally contaminated
with AFB1 and AFB2 on broilers in the starter period
were greater than those in the grower period, which
showed that young birds were more sensitive to afla-
toxin toxicity.
In conclusion, our results indicate that feeding corn
naturally contaminated with AFB1 and AFB2 slightly
changed serum biochemical parameters and impaired
hepatic tissues and hepatic antioxidant functions. This
influence was greater when broilers were exposed to
36.9 and 6.4 μg/kg of AFB1 and AFB2 in the starter
period and 95.2 and 17.0 μg/kg of AFB1 and AFB2 in
the grower period, respectively.
ACKNOWLEDGMENTS
We are grateful for the financial support provided by
the project “Feed safety and efficient use technology for
poultry,” the specific research supporting program for
academic sustentation research team in Sichuan Ag-
ricultural University and the safety of the mycotoxin
warning program of the Agricultural Ministry of China.
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